Rna-coded antibody

ABSTRACT

The present application describes an antibody-coding, non-modified or modified RNA and the use thereof for expression of this antibody, for the preparation of a pharmaceutical composition, in particular a passive vaccine, for treatment of tumours and cancer diseases, cardiovascular diseases, infectious diseases, autoimmune diseases, virus diseases and monogenetic diseases, e.g. also in gene therapy. The present invention furthermore describes an in vitro transcription method, in vitro methods for expression of this antibody using the RNA according to the invention and an in vivo method.

This application is a continuation of U.S. application Ser. No.15/007,072, filed Jan. 26, 2016, which is a continuation of U.S.application Ser. No. 13/709,897, filed Dec. 10, 2012, which is acontinuation of U.S. application Ser. No. 12/522,214, filed Jan. 4,2010, which is a national phase application under 35 U.S.C. §371 ofInternational Application No. PCT/EP2008/000081, filed Jan. 8, 2008,which claims benefit of German Application No. 10 2007 001 370.3, filedJan. 9, 2007, the entire contents of each of which are incorporatedherein by reference.

The present application describes an antibody-coding, non-modified ormodified RNA and the use thereof for expression of this antibody, forthe preparation of a pharmaceutical composition, in particular a passivevaccine, for treatment of tumours and cancer diseases, cardiovasculardiseases, infectious diseases, autoimmune diseases, virus diseases andmonogenetic diseases, e.g. also in gene therapy. The present inventionfurthermore describes an in vitro transcription method, in vitro methodsfor expression of this antibody using the RNA according to the inventionand an in vivo method.

The occurrence of tumours and cancer diseases is, alongsidecardiovascular and infectious diseases, one of the most frequent causesof death in modern societies and is associated with usually considerablecosts during the therapy and subsequent rehabilitation measures. Thetreatment of tumours and cancer diseases depends greatly, for example,on the nature of the tumour which occurs and at present conventionallyis undertaken by using radio- or chemotherapy, in addition to invasiveinterventions. However, these therapies represent an exceptional burdenon the immune system, and in some cases can be employed to only alimited extent. Furthermore, these therapy forms usually require longpauses between the individual treatments for regeneration of the immunesystem. In recent years, alongside these “conventional methods”,molecular biology programmes in particular have emerged as promising forthe treatment or for assisting these therapies.

An example of these molecular biology methods comprises the use ofantibodies or immunoglobulins as essential effectors of the immunesystem. Antibodies or immunoglobulins can be generated either in vitroby using known molecular biology methods or by the immune system of theorganism itself to be treated. The immune system of higher vertebratesthus has two separate functions of the immune system: the innate immunesystem, which reacts non-specifically to pathogens (e.g. bymacrophage-mediated phagocytosis) and the adaptive immune system, whichreacts specifically to pathogens by means of specialized effector cells(e.g. B and T cells). The antibodies or immunoglobulins which aresecreted by plasma cells during an immune response are part of thisadaptive immune system. Together with the complement system, they formthe humoral branch of the immune response.

Alongside their essential importance for the immune system in highervertebrates, precisely because of their high affinity and specificityfor a particular antigen antibodies are an outstanding means both inbiochemical and molecular biology research and in diagnostics andmedical uses. Thus, antibodies are capable of binding specifically totheir target structures (e.g. antigens, which substantially compriseproteins, peptides, in some cases lipids, carbohydrates etc.) and ofthereby blocking (inhibiting) or, where appropriate, labelling these.They can moreover activate the immune system by means of their Fc part,so that the labelled cells are destroyed. Over 100 therapeuticantibodies are currently to be found in clinical studies. Antibodieswhich can be employed in cancer therapy play by far the greatest role inthis context. Most of the antibodies prepared for this at present aremonoclonal antibodies which originate originally, for example, from themouse. In order to prevent an immune reaction against such monoclonalantibodies, at present chiefly humanized or human antibodies areemployed for therapy (cf. David Male; “Immunologie auf einen Blick[Immunology at a Glance]”, 1st German edition, 2005, Elsevier-Urban &Fischer Verlag; Charles A. Janeway, Paul Travers, Mark Walport and MarkShlomchik, Immunobiology, 5th edition, 2001, Garland Publishing;Dissertation by Christian Klein, Monoklonale Antikorper and rekombinanteAntikorperfragmente gegen sekundare Arzneipflanzenmetabolite [MonoclonalAntibodies and Recombinant Antibody Fragments Against SecondaryMedicinal Plant Metabolites], 2004; Andreas Schmiedl and Stefan Dübel,Rekombinante Antikorper & Phagen-Display [Recombinant Antibody & PhageDisplay], 2004, Molekulare Biotechnologie [Molecular Biotechnology](Wiley-VCH)).

Antibodies generally can be assigned to the group of immunoglobulins.These immunoglobulins can in turn be differentiated into five mainclasses of immunoglobulins on the basis of their heavy chain, the IgM(μ), IgD (δ), IgG (γ), IgA (α) and IgE (ε) antibodies, IgG antibodiesmaking up the largest proportion. Immunoglobulins can moreover bedifferentiated into the isotypes κ and λ on the basis of their lightchains.

In spite of their different specificity, antibodies are structurallyquite similar in construction. Thus, IgG antibodies typically are builtup two identical light and two heavy protein chains which are bonded toone another via disulfide bridges. The light chain comprises theN-terminal variable domain V_(L) and the C-terminal constant domainC_(L). The heavy chain of an IgG antibody can be divided into anN-terminal variable domain V_(H) and three constant domains C_(H)1,C_(H)2 and C_(H)3 (cf. FIG. 1). While the amino acid sequence is largelythe same in the region of the constant domains, wide differences insequence are typically found within the variable domains.

The antibody repertoire of a human comprises about at least 10¹¹different antibody specificities. In higher vertebrates, the formationof antibodies takes place naturally in the immune system by somaticrecombination. In this context, an organism is indeed theoreticallycapable of generating an antibody of appropriate specificity against anyantigen. However, if each of these antibodies were to be coded by anendogenous gene, they would burst the human genome. Instead, in humansantibody genes are composed of a large number of individual genesegments. The part of the antibody gene which codes for the variableregion of a light chain is formed from a V gene segment and a J genesegment. In this context, numerous different V and J segments areavailable, which can be combined with one another virtually as desired.In this context, the variable region of a heavy chain is composed ofthree different gene segments. In addition to the V and J segments,additional D segments are also found here. The V_(H), D_(H) and J_(H)segments can likewise be combined with one another virtually as desiredto form the variable region of the heavy chain (cf. FIG. 2). Themechanism by which the various gene segments are combined to formcomplete antibody genes is called immunoglobulin rearrangement orsomatic recombination. It takes place exclusively in B lymphocytes atcertain times of cell development.

In addition to pure gene rearrangement, further mechanisms forincreasing the antibody diversity also exist. Two mechanisms which areaccompanied by somatic recombination are first to be mentioned in thiscontext: The junctional diversity in this context describes controlledimprecise joining together of the rearranged gene segments, as a resultof which random removal and insertion of nucleotides occurs at thecleavage sites. A further combinatorial diversity results from thepossibility of combining a particular rearranged light chain with aparticular rearranged heavy chain. Finally, the diversity of antibodiesis also additionally increased after successful rearrangement and lateractivation of B cells, in that an affinity maturation of antibodiesoccurs due to an increased rate of mutation in the region of thevariable regions of activated B cells (somatic hypermutation).

In addition to the formation of antibodies which takes place naturallyby the immune system of the particular organism, antibodies can also begenerated by molecular biology methods. However, in order to utilize thesystem elaborated for specification of antibody formation andspecification thereof for particular antigens or nucleic acids, theformation of antibodies is at present typically induced in selectedorganisms by injection of a particular antigen, and the antibody is thenisolated from the organism for further use. In this context, the Blymphocytes of the organism are conventionally purified selectively andfused with an immortal myeloma cell to form a hybridoma cell. Thosecells which secrete the corresponding antigen-specific antibodies arethen determined by selection methods.

In addition to use of hybridoma cells, recombinant preparation of theseantibodies with the desired specificity is also possible after isolationand sequencing. Cells which provide the required posttranslationalmodifications are typically used for this. On the basis of the immunereaction with formation of human anti-mouse antibodies in the humanorganism in the case of native antibodies produced in the mouse (or inother hosts), chimeric, humanized or human antibodies are preferablyprepared here.

After expression, these antibodies, optionally prepared by recombinantmethods, can be employed as agents both in biochemical and molecularbiology research, and in diagnostics and for medical uses.

In medical uses, however, in many cases antibodies can be employeddirectly only with difficulty, since these usually have only a veryshort half-life in vivo and therefore, possibly, cannot reach theirtarget antigen or their target nucleic acid at all. This requires eitherhigh active compound concentrations of the desired antibody, oralternative methods which are suitable for providing large amounts ofantibodies in vivo.

Such methods include, e.g. molecular medicine methods of gene therapyand genetic vaccination which, when used generally in the therapy andprevention of diseases, have considerable effects on medical practice.Both methods are based on the introduction of nucleic acids into cellsor into tissue of the patient and on subsequent processing by the cellsor, respectively, tissue of the information coded by the nucleic acidsintroduced, i.e. expression of the desired polypeptides, e.g.antibodies, in the cells or respectively, the tissue.

The conventional procedure of methods of gene therapy and of geneticvaccination to date is based on the use of DNA to sluice the requiredgenetic information into the cell. Various methods for introducing DNAinto cells have been developed in this connection, such as, for example,calcium phosphate transfection, polyprene transfection, protoplastfusion, electroporation, microinjection, lipofection and the use of genecanons, lipofection in particular having emerged as a suitable method.

A further method which has been proposed in particular in the case ofgenetic vaccination methods is the use of DNA viruses as DNA vehicles.Such viruses have the advantage that because of their infectiousproperties a very high transfection rate can be achieved. The virusesused are genetically modified, so that no functional infectiousparticles are formed in the transfected cell. The use of DNA viruses asDNA vehicles, however, has been criticized in recent years because ofthe risk of recombination of non-active viruses to give active viruses.

The use of DNA as an agent in gene therapy and genetic vaccination orfor passive immunization (by passive vaccines), e.g. by using codingsequences for antibodies, may, however, also be less advantageous fromsome points of view. DNA is degraded only relatively slowly in thebloodstream, so that when (foreign) DNA is used as the coding sequencefor a desired protein, a formation of anti-DNA antibodies may occur,which has been confirmed in an animal model in the mouse (Gilkeson etal., J. Clin. Invest. 1995, 95: 1398-1402). The possible persistence of(foreign) DNA in the organism can thus lead to a hyperactivation of theimmune system, which as is known results in splenomegaly in mice(Montheith et al., Anticancer Drug Res. 1997, 12(5): 421-432).Furthermore, (foreign) DNA can interact with the host genome, and inparticular cause mutations by integration into the host genome. Thus,for example, the (foreign) DNA introduced may be inserted into an intactgene, which represents a mutation which can impede or even completelyswitch off the function of the endogenous gene. On the one hand enzymesystems which are vital for the cell may be destroyed by suchintegration events, and on the other hand there is also the danger of atransformation of the cell modified in this way into a degenerated stateif a gene which is decisive for regulation of cell growth is modified bythe integration of the foreign DNA. With the methods to date of genetherapy and genetic vaccination and also of passive immunization, a riskof development of cancer therefore cannot necessarily be ruled out when(foreign) DNA is used. In this context, passive immunization (byso-called “passive vaccines”) is to be strictly differentiated fromso-called active immunization. In active immunization, an antigen(“active vaccine”) is typically administered, after which the organismforms antibodies against this antigen. Active immunization thus createsa permanent immunization of the organism against the particular antigen,which can be associated with the disadvantages described above. Inpassive immunization, in contrast, only an antiserum or the purifiedantibody itself (“passive vaccine”) is administered to the organism. Thecoding sequence for the antibody can likewise be administered, asdescribed above, as a so-called passive vaccine for passiveimmunization.

Summarizing, in the prior art there is an increased demand for and aconsiderable interest in agents which are suitable for employingantibodies effectively in vivo, in particular for providing increasedactive compound amounts of antibodies in vivo, without the riskshitherto associated with the use of DNA.

This object is achieved according to the invention by the use of an RNA(sequence) for intracellular expression of an antibody, wherein the RNA(sequence) codes for an antibody or contains at least one coding region,which codes for at least one antibody, respectively. In connection withthe present invention, an antibody-coding RNA according to the inventionincludes any RNA which encodes an antibody. More generally, the RNA ofthe present invention (directed to intracellular expression) contains atleast one coding region, wherein the at least one coding region codesfor at least one antibody. If more than one coding region is containedin the RNA molecule of the invention, the second, third etc. codingregion may code for antibodies as well, which may be the same ordistinct from the first antibody coding region. In a preferredembodiment, the inventive RNA contains at least two coding regions, allof them coding for identical or distinct antibodies. In still anotherembodiment of the present invention, an inventive RNA may code for morethan one antibody within the same coding region. In summary, theinventive RNA may be mono-, bi- or multicistronic, codes for at leastone antibody.

The antibody-coding RNA according to the invention can besingle-stranded or double-stranded, linear or circular, or in particularin the form of mRNA. The antibody-coding RNA according to the inventionis particularly preferably in the form of single-stranded RNA, even morepreferably in the form of mRNA.

An antibody-coding RNA according to the invention preferably has alength of from 50 to 15,000 nucleotides, more preferably a length offrom 50 to 10,000 nucleotides, even more preferably a length of from 500to 10,000 nucleotides and most preferably a length of from 500 to 7,000,500 to 5,000 or 700 to 3,000 nucleotides.

In connection with the present invention, the antibodies coded by theRNA according to the invention can be chosen from all antibodies, e.g.from all antibodies which are generated by recombinant methods or arenaturally occurring and are known to a person skilled in the art fromthe prior art, in particular antibodies which are (can be) employed fortherapeutic purposes or for diagnostic or for research purposes or havebeen found with particular diseases, e.g. cancer diseases, infectiousdiseases etc.

In the context of the present invention, antibodies which are coded byan RNA according to the invention typically include all antibodies(described above) which are known to a person skilled in the art, e.g.naturally occurring antibodies or antibodies generated in a hostorganism by immunization, antibodies prepared by recombinant methodswhich have been isolated and identified from naturally occurringantibodies or antibodies generated in a host organism by (conventional)immunization or have been generated with the aid of molecular biologymethods, as well as chimeric antibodies, human antibodies, humanizedantibodies, bispecific antibodies, intrabodies, i.e. antibodiesexpressed in cells and possibly localized in particular cellcompartments, and fragments of the abovementioned antibodies. Insofar,the term antibody is to be understood in its broadest meaning. In thiscontext, antibodies in general typically comprise a light chain and aheavy chain, both of which have variable and constant domains. The lightchain comprises the N-terminal variable domain V_(L) and the C-terminalconstant domain C_(L). The heavy chain of an IgG antibody, in contrast,can be divided into an N-terminal variable domain V_(H) and threeconstant domains C_(H)1, C_(H)2 and C_(H)3 (cf. FIG. 1).

RNA molecules according to the invention can also be prepared on thebasis of polyclonal antibodies or, as an antibody-coding RNA cocktail,can have a polyclonal character. In the context of this invention,polyclonal antibodies are typically mixtures of antibodies against aspecific antigen or immunogen or epitope of a protein which have beengenerated by immunization of a host organism, for example mammals, i.e.animals, including cattle, pigs, dogs, cats, donkeys, monkeys, includingrodents, e.g. mice, hamsters, rabbits etc., and man. Polyclonalantibodies conventionally recognize different epitopes or regions of thesame specific antigen, each of these epitopes in turn being capable ofgenerating a clone of B lymphocytes which produces an antibody againstthis epitope. From such polyclonal antibodies or from the antibody seraobtained from the host organism, the individual antibodies specificagainst the particular epitopes can be obtained by individualization tomonoclonal antibodies. The present invention accordingly also providesRNA which codes for a monoclonal antibody obtained by individualizationof polyclonal antibodies.

Monoclonal antibodies in the context of this invention are thereforetypically antibodies which are specific for a particular antigen orepitope (of a protein), i.e. bind this antigen or epitope (of a protein)with a high affinity, and conventionally are expressed by a hybridomacell. For the preparation of such monoclonal antibodies, thecorresponding antigen or immunogen or epitope of a protein is typicallyinjected at least once, but typically several times, into a hostorganism as described here, as a result of which the immune system ofthe host organism, in the presence of suitable adjuvants, is preferablystimulated to antibody production via activation of correspondinglyspecific B cells. The B lymphocytes are then conventionally selectivelypurified from the spleen or other organs or fluids suitable for thisfrom an animal immunized in this manner, and are fused with an immortalmyeloma cell to give the so-called hybridoma cell. After selectionmethods and cloning of the hybridomas or hybridoma cells formed, thoseclones which secernate, i.e. express and secrete, antibodies of thedesired specificity can be determined. These clones can be isolated andsequenced with known molecular biology methods. The data obtained fromsuch a sequencing can serve further in a nucleic acid synthesis forgeneration of synthetic DNA sequences or for screening a cDNA libraryand isolation of the cDNA fragments and generation of a DNA or nucleicacid template for in vitro or in vivo synthesis of the RNA according tothe invention which codes for an antibody. Where appropriate, the RNAcontained in the hybridomas can also be isolated, for example byfractionation, and subsequently the RNA molecules according to theinvention which code for the hybridoma antibody can be purified bymethods known to the person skilled in the art.

Nevertheless, RNA molecules which code for non-human monoclonal orpolyclonal antibody, e.g. murine monoclonal antibodies or monoclonalantibodies from other, as described here, non-human host organisms orhybridoma cells are of only limited suitability for therapeutic use inhumans, since in the human organism itself they conventionally cause animmune reaction with formation of human anti-antibodies directed againstthese non-human host antibodies. As a result, such non-human monoclonalor polyclonal antibodies as a rule can be administered to a person onlya single time. To by-pass this problem, RNA molecules which code forchimeric, humanized and human antibodies can also be provided accordingto the invention.

Chimeric antibodies in the context of the present invention arepreferably antibodies in which the constant domains of an antibody asdescribed here have been replaced by human sequences. Preferably,chimeric antibodies are formed from monoclonal or polyclonal antibodiesas described here.

Humanized antibodies in the context of the present invention areantibodies in which the constant and variable domains described above ofthe non-human monoclonal or polyclonal antibodies, with the exception ofthe hypervariable regions, have been replaced by human sequences.

RNA molecules which code for human antibodies, i.e. antibodies whichhave completely human sequences, that is to say in the constant andvariable domains, including the hypervariable regions, can furthermorebe used in the context of the present invention. Such RNA moleculeswhich code for human antibodies can be isolated from human tissue ororiginate from immunized host organisms as described here, e.g. mice,which are then transgenic for the human IgG gene locus. RNA moleculeswhich code for human antibodies and have been isolated by means of phagedisplay and cloned with the aid of molecular biology methods arefurthermore provided (see below).

Antibodies which are coded by RNAs according to the inventionparticularly preferably include so-called full length antibodies, i.e.antibodies which comprise both the complete heavy and the complete lightchains, as described above.

RNAs which alternatively code for one or more antibody fragment(s) ofthe antibodies described above, instead of the corresponding full lengthantibody, can furthermore be provided in the context of the presentinvention. Examples of such antibody fragments are any antibodyfragments known to a person skilled in the art, e.g. Fab, Fab′, F(ab′)₂,Fc, Facb, pFc′, Fd, and Fv fragments of the abovementioned antibodiesetc. A diagram of the structure of such antibody fragments is shown byway of example in FIG. 4. Protein fragments consisting of the minimalbinding subunit of antibodies known as single-chain antibodies (scFvs)have excellent binding specificity and affinity for their ligands. Incontrast to antibodies, scFvs lack the non-binding regions. Accordingly,RNA encoding scFvs are also encompassed by the present invention.

For example, an Fab (fragment antigen binding) fragment typicallycomprises the variable and a constant domain of a light and a heavychain, e.g. the C_(H)1 and the V_(H) domain of the heavy chain and thecomplete light chain. The two chains are bonded to one another via adisulfide bridge. An Fab fragment thus conventionally contains thecomplete antigen-binding region of the original antibody and usually hasthe same affinity for the antigen, the immunogen or an epitope of aprotein. Antibody fragments, as also described above for antibodies, canbe prepared with the aid of molecular biology methods. In this context,the DNA sequences which code for the various domains of the antibodyfragment are cloned into a specific expression vector. The RNA whichcodes for these antibody fragments can then be expressed e.g. insuitable host cells. Suitable host cells in connection with the presentinvention include, inter alia, E. coli, yeasts, transgenic plants ormammalian cells etc. (see below). In contrast, an scFv fragment (singlechain variable fragment) typically comprises the variable domain of thelight and of the heavy chain, which are bonded to one another via anartificial polypeptide linker. In the cloning of such scFv fragments,RNAs which code for a V_(H) and V_(L), these being linked to one anotherby a polypeptide linker, are preferably provided. As a rule, apolypeptide built up from 15-25 glycine, proline and/or serine residues(cf. FIG. 5) or the associated nucleotide sequence is used at the RNAlevel for the provision of this component.

Furthermore, RNA molecules which code for bispecific antibodies can alsobe provided in the context of the present invention. Bispecificantibodies in the context of the present invention are preferablyantibodies which can act as adaptors between an effector and acorresponding target, e.g. for recruiting effector molecules (e.g.toxins, active compounds (drugs), cytokines etc.), targeting of effectorcells (e.g. CTL, NK cells, macrophages, granulocytes etc. (see, forexample, review by Kontermann R. E., Acta Pharmacol. Sin, 2005, 26(1):1-9). In this context, bispecific antibodies are in principle built upsuch as is described here in general for antibodies, these bispecificantibodies e.g. recognizing two different antigens, immunogens orepitopes, or active compounds, cells, or other molecules (or structures)as mentioned above, i.e. the antigen-binding regions of the antibody arespecific for two different molecules (or structures). The variousantigens, immunogens or epitopes etc., for example, can thus be broughtspatially close. Furthermore, by the binding e.g. of a binding domain orother specificities, the function of the antibody can be extendedspecifically, e.g. of a binding protein, an immunotoxin etc. Suchbispecific antibodies can also be single-chain antibodies (e.g. scFvfragments etc.). Bispecific antibodies can be used, for example, tobring two reaction partners, e.g. two cells, two proteins, a protein andthe substrate thereof etc., spatially close in order to promote aninteraction between these (e.g. protein-protein interactions, substrateconversions, modifications etc.). Bispecific antibodies are used aboveall to bring effector cells (such as, for example, T cells, NK cells,macrophages etc.) and target cells (e.g. tumour cells, infected cellsetc.) spatially close. Examples of bispecific antibodies can include,without being limited thereto, e.g. those antibodies or antibodyfragments which bind on the one hand a surface factor as described here,and on the other hand an antigen as described here, preferably a tumourantigen as described here. This includes e.g. CD28 and a tumour antigen(Grosse-Hovest L. et al., 2003, Eur. Immunol. 33(5); 1334-40, (Arecombinant bispecific single-chain antibody induces targeted,supra-agonistic CD28-stimulation and tumor cell killing)), CD19 and CD3(CD19 tumour antigen of B cell lymphoma) etc.

Without being limited thereto, according to the present invention RNAswhich code for antibodies inter alia code for those antibodies whichbind antigens or specific nucleic acids. Antigens in the context of thepresent invention are typically molecules which are recognized asexogenous by the immune system and conventionally cause an immunereaction or immune response with the formation of antibodies directedspecifically against them. However, antigens can also include,especially in the case of autoimmune diseases, endogenous molecules orstructures which are incorrectly recognized as exogenous by the immunesystem and thereby trigger an immune reaction. Alternatively formulated,antigens are therefore all molecules which are recognized by an antibodyin the context of the present invention. Antigens substantially compriseproteins, peptides or epitopes of these proteins or peptides. In thiscontext, epitopes (also called “antigenic determinants”) are typicallysmall regions (molecular sections) lying on the surface of such proteinor peptide structures and having a length of from 5 to 15, in rare casealso to 25, preferably 6 to 9 amino acids. Antigens can furthermore alsoinclude lipids, carbohydrates etc. In the context of the presentinvention, antigens also include, for example, so-called immunogens,i.e. antigens which lead to an immunity of the organism transfectedtherewith. Antigens by way of example include, without being limitedthereto, surface antigens of cells, tumour antigens etc. For example,according to the present invention antibodies can bind the followingantigens (which typically occur in vertebrates), e.g. tumour-specificsurface antigens (TSSA), e.g. 5T4, α5β1-integrin, 707-AP, AFP, ART-4,B7H4, BAGE, β-catenin/m, Bcr-abl, MN/C IX-antigen, CA125, CAMEL, CAP-1,CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD30, CD33, CD52, CD56,CD80, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam,ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2/neu, HLA-A*0201-R170I,HPV-E7, HSP70-2M, HAST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5,KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/Melan-A, MART-2/Ski, MC1R,myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NY-ESO1, PAP, proteinase-3, p190minor bcr-abl, Pml/RARα, PRAIVIE, PSA, PSM, PSMA, RAGE, RU1 or RU2,SAGE, SART-1 or SART-3, survivin, TEL/AML1, TGFβ, TPI/m, TRP-1, TRP-2,TRP-2/INT2, VEGF and WT1, or sequences, such as e.g. NY-Eso-1 orNY-Eso-B. Tumour antigens can, for example, typically be responsible formetastasing, that is to say dissolving of tumour cells out of theirnative tissue, transfer into the vascular system (lymph or blood vesselsystem), exit from the vascular system and colonization in a new tissue.In this context, such tumour antigens which cause modified cell-cellinteractions compared with the native state are of interest inparticular.

Antibodies encoded by the inventive RNA may also be directed againsttumour antigens listed by Table 1 or Table 2. In particular, RNAencoding those antibodies may be used to treat (or, may be used toprepare a medicament to treat, respectively) the cancer diseases givenin the last column of Tables 1 and 2.

TABLE 1 Antigens expressed in cancer diseases Tumor antigen Name oftumor antigen Cancers or cancer diseases related thereto 5T4 colorectalcancer, gastric cancer, ovarian cancer 707-AP 707 alanine prolinemelanoma 9D7 renal cell carcinoma AFP alpha-fetoprotein hepatocellularcarcinoma, gallbladder cancer, testicular cancer, ovarian cancer,bladder cancer AlbZIP HPG1 prostate cancer alpha5beta1- Integrinalpha5beta6- colon cancer Integrin alpha-methylacyl- prostate cancercoenzyme A racemase ART-4 adenocarcinoma antigen lung cancer, head andneck cancer, recognized by T cells 4 leukemia, esophageal cancer,gastric cancer, cervical cancer, ovarian cancer, breast cancer, squamouscell carcinoma B7H4 ovarian cancer BAGE-1 B antigen bladder cancer, headand neck cancer, lung cancer, melanoma, squamous cell carcinoma BCL-2leukemia BING-4 melanoma CA 15-3/CA 27-29 breast cancer, ovary cancer,lung cancer, prostate cancer CA 19-9 gastric cancer, pancreatic cancer,liver cancer, breast cancer, gallbladder cancer, colon cancer, ovarycancer, lung cancer CA 72-4 ovarian cancer CA125 ovarian cancer,colorectal cancer, gastric cancer, liver cancer, pancreatic cancer,uterus cancer, cervix carcinoma, colon cancer, breast cancer, lungcancer calreticulin bladder cancer CAMEL CTL-recognized antigen onmelanoma melanoma CASP-8 caspase-8 head and neck cancer cathepsin Bbreast cancer cathepsin L breast cancer CD19 B-cell malignancies CD20CD22 CD25 CD30 CD33 CD4 CD52 CD55 CD56 CD80 CEA carcinoembryonic antigengut carcinoma, colorectal cancer, colon cancer, hepatocellular cancer,lung cancer, breast cancer, thyroid cancer, pancreatic cancer, livercancer cervix cancer, bladder cancer, melanoma CLCA2 calcium-activatedchloride lung cancer channel-2 CML28 leukemia Coactosin-like pancreaticcancer protein Collagen XXIII prostate cancer COX-2 ovarian cancer,breast cancer, colorectal cancer CT-9/BRD6 bromodomain testis-specificprotein Cten C-terminal tensin-like protein prostate cancer cyclin B1cyclin D1 ovarian cancer cyp-B cyclophilin B bladder cancer, lungcancer, T-cell leukemia, squamous cell carcinoma, CYPB1 cytochrom P4501B1 leukemia DAM-10/MAGE-B1 differentiation antigen melanoma, skintumors, ovarian cancer, melanoma 10 lung cancer DAM-6/MAGE-B2differentiation antigen melanoma, skin tumors, ovarian cancer, melanoma6 lung cancer EGFR/Her1 lung cancer, ovarian cancer, head and neckcancer, colon cancer, pancreatic cancer, breast cancer EMMPRIN tumorcell-associated extracellular lung cancer, breast cancer, bladder matrixmetalloproteinase inducer/ cancer, ovarian cancer, brain cancer,lymphoma EpCam epithelial cell adhesion molecule ovarian cancer, breastcancer, colon cancer, lung cancer EphA2 ephrin type-A receptor 2 gliomaEphA3 ephrin type-A receptor 2 melanoma, sarcoma, lung cancer ErbB3breast cancer EZH2 (enhancer of Zeste homolog 2) endometrium cancer,melanoma, prostate cancer, breast cancer FGF-5 fibroblast growthfactor-5 renal cell carcinoma, breast cancer, prostate cancer FNfibronectin melanoma Fra-1 Fos-related antigen-1 breast cancer,esophageal cancer, renal cell carcinoma, thyroid cancer G250/CAIXglycoprotein 250 leukemia, renal cell carcinoma, head and neck cancer,colon cancer, ovarian cancer, cervical cancer GAGE-1 G antigen 1 bladdercancer, lung cancer, sarcoma, melanoma, head and neck cancer GAGE-2 Gantigen 2 bladder cancer, lung cancer, sarcoma, melanoma, head and neckcancer GAGE-3 G antigen 3 bladder cancer, lung cancer, sarcoma,melanoma, head and neck cancer GAGE-4 G antigen 4 bladder cancer, lungcancer, sarcoma, melanoma, head and neck cancer GAGE-5 G antigen 5bladder cancer, lung cancer, sarcoma, melanoma, head and neck cancerGAGE-6 G antigen 6 bladder cancer, lung cancer, sarcoma, melanoma, headand neck cancer GAGE-7b G antigen 7b bladder cancer, lung cancer,sarcoma, melanoma, head and neck cancer GAGE-8 G antigen 8 bladdercancer, lung cancer, sarcoma, melanoma, head and neck cancer GDEP genedifferentially expressed prostate cancer in prostate GnT-VN-acetylglucosaminyltransferase V glioma, melanoma gp100 glycoprotein100 kDa melanoma GPC3 glypican 3 hepatocellular carcinoma, melanoma HAGEhelicase antigen bladder cancer HAST-2 human signet ring tumor-2 hepsinprostate Her2/neu/ErbB2 human epidermal receptor-2/ breast cancer,bladder cancer, melanoma, neurological ovarian cancer, pancreas cancer,gastric cancer HERV-K-MEL melanoma HNE human neutrophil elastaseleukemia homeobox NKX prostate cancer 3.1 HOM-TES-14/ ovarian cancerSCP-1 HOM-TES-85 HPV-E6 cervical cancer HPV-E7 cervical cancer HST-2gastric cancer hTERT human telomerase reverse breast cancer, melanoma,lung cancer, transcriptase ovarian cancer, sarcoma, Non-Hodgkin-lymphoma, acute leukemia iCE intestinal carboxyl esterase renal cellcarcinoma IGF-1R colorectal cancer IL-13Ra2 interleukin 13 receptoralpha glioblastoma 2 chain IL-2R colorectal cancer IL-5 immature lamininrenal cell carcinoma receptor kallikrein 2 prostate cancer kallikrein 4prostate cancer Ki67 prostate cancer, breast cancer, Non-Hodgkin-lymphoma, melanoma KIAA0205 bladder cancer KK-LC-1 Kita-kyushulung cancer antigen lung cancer 1 KM-HN-1 tongue cancer, hepatocellularcarcinomas, melanoma, gastric cancer, esophageal, colon cancer,pancreatic cancer LAGE-1 L antigen bladder cancer, head and neck cancer,melanoma livin bladder cancer, melanoma MAGE-A1 melanoma antigen-A1bladder cancer, head and neck cancer, melanoma, colon cancer, lungcancer, sarcoma, leukemia MAGE-A10 melanoma antigen-A10 bladder cancer,head and neck cancer, melanoma, colon cancer, lung cancer, sarcoma,leukemia MAGE-A12 melanoma antigen-A12 bladder cancer, head and neckcancer, melanoma, colon cancer, lung cancer, sarcoma, leukemia, prostatecancer, myeloma, brain tumors MAGE-A2 melanoma antigen-A2 bladdercancer, head and neck cancer, melanoma, colon cancer, lung cancer,sarcoma, leukemia MAGE-A3 melanoma antigen-A3 bladder cancer, head andneck cancer, melanoma, colon cancer, lung cancer, sarcoma, leukemiaMAGE-A4 melanoma antigen-A4 bladder cancer, head and neck cancer,melanoma, colon cancer, lung cancer, sarcoma, leukemia MAGE-A6 melanomaantigen-A6 bladder cancer, head and neck cancer, melanoma, colon cancer,lung cancer, sarcoma, leukemia MAGE-A9 melanoma-antigen-A9 bladdercancer, head and neck cancer, melanoma, colon cancer, lung cancer,sarcoma, leukemia MAGE-B1 melanoma-antigen-B1 melanoma MAGE-B10melanoma-antigen-B10 melanoma MAGE-B16 melanoma-antigen-B16 melanomaMAGE-B17 melanoma-antigen-B17 melanoma MAGE-B2 melanoma-antigen-B2melanoma MAGE-B3 melanoma-antigen-B3 melanoma MAGE-B4melanoma-antigen-B4 melanoma MAGE-B5 melanoma-antigen-B5 melanomaMAGE-B6 melanoma-antigen-B6 melanoma MAGE-C1 melanoma-antigen-C1 bladdercancer, melanoma MAGE-C2 melanoma-antigen-C2 melanoma MAGE-C3melanoma-antigen-C3 melanoma MAGE-D1 melanoma-antigen-D1 melanomaMAGE-D2 melanoma-antigen-D2 melanoma MAGE-D4 melanoma-antigen-D4melanoma MAGE-E1 melanoma-antigen-E1 bladder cancer, melanoma MAGE-E2melanoma-antigen-E2 melanoma MAGE-F1 melanoma-antigen-F1 melanomaMAGE-H1 melanoma-antigen-H1 melanoma MAGEL2 MAGE-like 2 melanomamammaglobin A breast cancer MART-1/Melan-A melanoma antigen recognizedby melanoma T cells-1/melanoma antigen A MART-2 melanoma antigenrecognized by melanoma T cells-2 matrix protein 22 bladder cancer MC1Rmelanocortin 1 receptor melanoma M-CSF macrophage colony-stimulatingovarian cancer factor gene mesothelin ovarian cancer MG50/PXDN breastcancer, glioblastoma, melanoma MMP 11 M-phase phosphoprotein 11 leukemiaMN/CA IX- renal cell carcinoma antigen MRP-3 multidrugresistance-associated lung cancer protein 3 MUC1 mucin 1 breast cancerMUC2 mucin 2 breast cancer, ovarian cancer, pancreatic cancer NA88-A NAcDNA clone of patient M88 melanoma N-acetylglucosaminyl- transferase-VNeo-PAP Neo-poly(A) polymerase NGEP prostate cancer NMP22 bladder cancerNPM/ALK nucleophosmin/anaplastic lymphoma kinase fusion protein NSEneuron-specific enolase small cell cancer of lung, neuroblastoma, Wilm'tumor, melanoma, thyroid cancer, kidney cancer, testicle cancer,pancreas cancer NY-ESO-1 New York esophageous 1 bladder cancer, head andneck cancer, melanoma, sarcoma, B-lymphoma, hepatoma, pancreatic cancer,ovarian cancer, breast cancer NY-ESO-B OA1 ocular albinism type 1protein melanoma OFA-iLRP oncofetal antigen-immature laminin leukemiareceptor OGT O-linked N-acetylglucosamine transferase gene OS-9osteocalcin prostate cancer osteopontin prostate cancer, breast cancer,ovarian cancer p15 protein 15 p15 melanoma p190 minor bcr- abl p53PAGE-4 prostate GAGE-like protein-4 prostate cancer PAI-1 plasminogenacitvator inhibitor 1 breast cancer PAI-2 plasminogen acitvatorinhibitor 2 breast cancer PAP prostate acic phosphatase prostate cancerPART-1 prostate cancer PATE prostate cancer PDEF prostate cancerPim-1-Kinase Pin1 Propyl isomerase prostate cancer POTE prostate cancerPRAME preferentially expressed antigen melanoma, lung cancer, leukemia,head of melanoma and neck cancer, renal cell carcinoma, sarcoma prosteinprostate cancer proteinase-3 PSA prostate-specific antigen prostatecancer PSCA prostate cancer PSGR prostate cancer PSM PSMAprostate-specific membrane prostate cancer antigen RAGE-1 renal antigenbladder cancer, renal cancer, sarcoma, colon cancer RHAMM/CD168 receptorfor hyaluronic acid leukemia mediated motility RU1 renal ubiquitous 1bladder cancer, melanoma, renal cancer RU2 renal ubiquitous 1 bladdercancer, melanoma, sarcoma, brain tumor, esophagel cancer, renal cancer,colon cancer, breast cancer S-100 melanoma SAGE sarcoma antigen SART-1squamous antigen rejecting tu- esophageal cancer, head and neck cancer,mor 1 lung cancer, uterine cancer SART-2 squamous antigen rejecting tu-head and neck cancer, lung cancer, renal mor 1 cell carcinoma, melanoma,brain tumor SART-3 squamous antigen rejecting tu- head and neck cancer,lung cancer, mor 1 leukemia, melanoma, esophageal cancer SCC squamouscell carcinoma antigen lung cancer Sp17 sperm protein 17 multiplemyeloma SSX-1 synovial sarcoma X breakpoint 1 hepatocellular cellcarcinom, breast cancer SSX-2/HOM-MEL-40 synovial sarcoma X breakpoint 2breast cancer SSX-4 synovial sarcoma X breakpoint 4 bladder cancer,hepatocellular cell carcinoma, breast cancer STAMP-1 prostate cancerSTEAP six transmembrane epithelial prostate cancer antigen prostatesurvivin bladder cancer survivin-2B intron 2-retaining survivin bladdercancer TA-90 melanoma TAG-72 prostate carcinoma TARP prostate cancerTGFb TGFbeta TGFbRII TGFbeta receptor II TGM-4 prostate-specificprostate cancer transglutaminase TRAG-3 taxol resistant associatedbreast cancer, leukemia, and melanoma protein 3 TRG testin-related geneTRP-1 tyrosine related protein 1 melanoma TRP-2/6b TRP-2/novel exon 6bmelanoma, glioblastoma TRP-2/INT2 TRP-2/intron 2 melanoma, glioblastomaTrp-p8 prostate cancer Tyrosinase melanoma UPA urokinase-typeplasminogen breast cancer activator VEGF vascular endothelial growthfactor VEGFR-2/FLK-1 vascular endothelial growth factor receptor-2 WT1Wilm' tumor gene gastric cancer, colon cancer, lung cancer, breastcancer, ovarian cancer, leukemia

TABLE 2 Mutant antigens expressed in cancer diseases Mutant antigen Nameof mutant antigen Cancers or cancer diseases related theretoalpha-actinin-4/m lung carcinoma ARTC1/m melanoma bcr/abl breakpointcluster region- CML Abelson fusion protein beta-Catenin/m beta-Cateninmelanoma BRCA1/m breast cancer BRCA2/m breast cancer CASP-5/m colorectalcancer, gastric cancer, endometrial carcinoma CASP-8/m head and neckcancer, squamous cell carcinoma CDC27/m cell-division-cycle 27 CDK4/mcyclin-dependent kinase 4 melanoma CDKN2A/m melanoma CML66 CML COA-1/mcolorectal cancer DEK-CAN fusion protein AML EFTUD2/m melanoma ELF2/mElongation factor 2 lung squamous cell carcinoma ETV6-AML1 Ets variantgene6/acute myeloid ALL leukemia 1 gene fusion protein FN1/m fibronectin1 melanoma GPNMB/m melanoma HLA-A*0201- arginine to isoleucine exchangerenal cell carcinoma R170I at residue 170 of the alpha-helix of thealpha2-domain in the HLA-A2 gene HLA-A11/m melanoma HLA-A2/m renal cellcarcinoma HSP70-2M heat shock protein 70-2 mutated renal cell carcinoma,melanoma, neuroblastoma KIAA0205/m bladder tumor K-Ras/m pancreaticcarcinoma, colorectal carcinoma LDLR-FUT LDR-Fucosyltransferase fusionmelanoma protein MART2/m melanoma ME1/m non-small cell lung carcinomaMUM-1/m melanoma ubiquitous mutated 1 melanoma MUM-2/m melanomaubiquitous mutated 2 melanoma MUM-3/m melanoma ubiquitous mutated 3melanoma Myosin class I/m melanoma neo-PAP/m melanoma NFYC/m lungsquamous cell carcinoma N-Ras/m melanoma OGT/m colorectal carcinomaOS-9/m melanoma p53/m Pml/RARa promyelocytic leukemia/retinoic APL, PMLacid receptor alpha PRDX5/m melanoma PTPRK/m receptor-typeprotein-tyrosine melanoma phosphatase kappa RBAF600/m melanoma SIRT2/mmelanoma SYT-SSX-1 synaptotagmin I/synovial sarcoma sarcoma X fusionprotein SYT-SSX-2 synaptotagmin I/synovial sarcoma sarcoma X fusionprotein TEL-AML1 translocation Ets-family AML leukemia/acute myeloidleukemia 1 fusion protein TGFbRII TGFbeta receptor II colorectalcarcinoma TPI/m triosephosphate isomerase melanoma

In a preferred embodiment according to the invention, antibodies encodedby the inventive RNA are directed against the following (protein)antigens (whereby the RNA molecules may be used for the preparation of amedicament, e.g. a pharmaceutical composition or more preferably a(passive) vaccine in the meaning of the present invention), are selectedfrom the group consisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1,alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m,alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE1,BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsinB, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55,CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66,COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6, Cten,cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEKCAN, EFTUD2/m,EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2,FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin,Her2/neu, HERV-K-MEL, HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE,homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M,HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature lamininreceptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m,KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3,MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1,MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1,MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrixprotein 22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP 11, MN/CAIX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin classI/m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m,NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1,OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PART-1, PATE, PDEF,Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAIVIE, PRDX5/m, prostein,proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m,RHAMM/CD168, RU1, RU2, S-100, SAGE, SART1, SART-2, SART-3, SCC, SIRT2/m,Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP, survivin,survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1,TGFbeta, TGFbetaRTI, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b,TRP/INT2, TRP-p8, tyrosinase, UPA, VEGF, VEGFR-2/FLK-1, and WT1.

In a particularly preferred embodiment, the RNA codes for antibodieswhich are directed against protein antigens selected from the groupconsisting of MAGE-A1, MAGE-A6, melan-A, GP100, tyrosinase, survivin,CEA, Her-2/neu, WT1, PRAIVIE, EGFRI (epidermal growth factor receptor1), mucin-1 and SEC61G, hTERT, 5T4, NY-Esol, and TRP-2, more preferablyfrom sequences of group consisting of MAGE-A1 [accession number M77481],MAGE-A6 [accession number NM_005363], melan-A [accession numberNM_005511], GP100 [accession number M77348], tyrosinase [accessionnumber NM_000372], survivin [accession number AF077350], CEA [accessionnumber NM_004363], Her-2/neu [accession number M11730], WT1 [accessionnumber NM_000378], PRAME [accession number NM_006115], EGFRI (epidermalgrowth factor receptor 1) [accession number AF288738], mucin-1[accession number NM_002456] and SEC61G [accession number NM_014302],hTERT [accession number NM_198253], 5T4 [accession number NM_006670],NY-Esol [accession number NM_001327], and TRP-2 [accession numberNM_001922].

Antibodies (and therefore also the RNAs according to the invention onwhich these antibodies are based) which bind the antigens described hereand, possibly, other antigens or nucleic acids can be identified e.g. bymeans of the method of phage display developed by George P. Smith. Inthis context, antibodies or antibody fragments are typically expressedon the surface of filamentous phages (Smith, G. P., 1985, “Filamentousfusion phage: novel expression vectors that display cloned antigens onthe virion surface”, Science 228; 1315-1317). For this there areconventionally 3 to 5 copies of the surface protein gpIII on theproximal end of the phage, with the aid of which the phage infectsbacteria cells via the F pilus thereof. In phage display, for example,the DNA for an antibody fragment which codes the antigen-bindingvariable domain is then cloned in-frame before the gpIII gene. Inprotein biosynthesis, a fusion protein is formed therefrom, which isexpressed on the virus surface without the phage losing itsinfectiousness. With the aid of the phage display technique, it ispossible to generate large antibody libraries, each phage expressing adifferent antibody fragment on the surface. To this extent, theunderlying RNA is therefore also available. A particular antibodyfragment can be isolated from such a library by a method called “phagepanning”. For this, the corresponding antigen is bound to a matrix andincubated with the phage suspension. The phages which present anappropriate antibody fragment interact with the fixed antigen, while theother phages are removed by a washing step. The phages isolated aremultiplied, for example, in E. coli. The DNA is isolated accordingly andthe gene sequence is determined. Expression constructs which contain thecDNA coding for the entire antibody or antibody fragments can then bedeveloped with the aid of genetic engineering methods. An RNA (mRNA)which codes for the antibody can be generated from this cDNA by means ofin vitro transcription (see below). Nucleic acids or, respectively, mRNAcoding for monoclonal antibodies which are entirely of human origin areobtained in this manner.

In the context of the present invention, RNA according to the inventionwhich codes for antibodies as described above is also suitable forcoding so-called intrabodies or for rendering possible an expression ofintrabodies. Intrabodies in the context of the present invention caninclude any of the antibodies or antibody fragments described here.Intrabodies are intracellularly expressed antibodies, i.e. antibodieswhich are coded by nucleic acids localized in the cell and are expressedthere. For this, an RNA according to the invention which encodes theantibodies or antibody fragments as described above is introduced intocells beforehand, for example with the aid of transfection methodsaccording to the invention or other suitable transfection methods (seebelow) and, where appropriate, thereafter transplanted into an organismor being or introduced directly as nucleic acids into an organism orbeing. In this context (irrespective of whether an an intrabody or asecreted antibody shall be introduce into the cell), the RNA accordingto the invention (or a corresponding nucleic acid) can be introduced inthe naked form or as a complex with suitable carriers (e.g. liposomes)into the organism or the being or can have such modifications (of theRNA) which, where appropriate together with one of the transfectionmethods mentioned, lead to a better cell uptake, e.g. any of the RNAmodifications mentioned here, such as, for example, lipid modificationsof the RNA according to the invention. An organism or a being inconnection with the present invention typically means mammals, i.e.animals, including cattle, pig, dog, cat, donkey, monkey, rodents, e.g.mouse, hamster, rabbit etc., and humans. Intrabodies can be localizedand expressed at certain sites in the cell. For example, intrabodies canbe expressed in the cytoplasm, the formation of disulfide bridgesusually being decreased under the reducing conditions of the cytoplasm.It has been possible to demonstrate, however, that cytoplasmicintrabodies, and in particular scFv fragments, can be functional.Cytoplasmic expression by the RNA according to the invention opens upthe possibility of also inhibiting cytoplasmic proteins. This is notpossible with treatment with monoclonal antibodies from the prior art,since these antibodies can reach only secreted and membrane-located(extracellular) proteins due to their secretion from the cell afterintracellular expression (which represents the major difference betweenantibodies and intrabodies). By expression of a signal peptide,intrabodies can be transported into the endoplasmic reticulum (ER) andthen secreted as with regular antibodies. In this case, typically onlysecreted or membrane-located proteins are a target for these antibodies.By additional coding of a C-terminal ER retention signal (for exampleKDEL (SEQ ID NO: 18)) by the RNA according to the invention, theintrabody can remain in the ER (where it may bind to specific antigenlocated in the ER) and prevent secretion of its antigen and/or transportof its antigen or its target molecule to the plasma membrane. Dependingon the requirement, intrabodies can include full length antibodies orantibody fragments as described above. Intrabodies in the context of thepresent invention preferably initially include full length antibodies,which are retained in the cell and not secreted from the cell (bywhatever technique, e.g. retention signal sequences etc.). However, ife.g. intracellular expression of full length antibodies is technicallynot possible or not appropriate, antibody fragments as described abovecan also be employed as intrabodies.

Antibodies which are coded by the RNA according to the inventionfurthermore also include those antibodies or antibody fragments whichhave a sequence identity to one of the antibodies or antibody fragmentsdescribed here of at least 70%, 80% or 85%, preferably at least 90%,more preferably at least 95% and most preferably at least 99% over theentire length of the coding nucleic acid or amino acid sequence of anantibody or antibody fragment as described here. Preferably, suchantibodies or antibody fragments have the same biological function asor, respectively, the specific activity of the corresponding full lengthantibody, e.g. the specific binding of particular antigens or nucleicacids. Accordingly, it is preferred, if the hypervariable region(s) areconserved or are modified by merely conservative mutations.

The biological function of antibodies described here which are coded bythe RNA according to the invention includes e.g. neutralization ofantigens, complement activation or opsonization. In the case ofneutralization of antigens, the antibody can bind to an antigen andthereby neutralize this. The antibody is conventionally blocked by thebinding of the antigen, and can therefore display its action onlyagainst one antigen, or two antigens in the case of bispecificantibodies. scFv antibody fragments are suitable above all for this(neutralization) function of an antibody, since they do not include thefunctions of the constant domains of an antibody. In the case ofcomplement activation, the complex system of complement proteins whichare dependent upon the Fc part of the antibody can be activated viabinding of antibodies. End products of the complement cascade typicallylead to lysis of cells and to the creation of a phlogistic(inflammatory) milieu. In the case of opsonization, pathogens or foreignparticles are rendered accessible to phagocytes by binding by anantibody via the constant domains of the antibody. Alternatively, theopsonized cells, which are recognized as foreign, can be lysed via anantibody-dependent, cell-mediated cytotoxicity (ADCC). In this context,NK cells in particular can perform lytic functions in this manner viaactivation of their Fc receptors.

In connection with the present invention, the term “identity” means thatthe sequences are compared with one another as follows. In order todetermine the percentage identity of two nucleic acid sequences, thesequences can first be aligned with respect to one another in ordersubsequently to make a comparison of these sequences possible. For thise.g. gaps can be inserted into the sequence of the first nucleic acidsequence and the nucleotides can be compared with the correspondingposition of the second nucleic acid sequence. If a position in the firstnucleic acid sequence is occupied by the same nucleotide as is the caseat a position in the second sequence, the two sequences are identical atthis position. The percentage identity between two sequences is afunction of the number of identical positions divided by the number ofall the positions compared in the sequences investigated. If e.g. aspecific sequence identity is assumed for a particular nucleic acid(e.g. a nucleic acid which codes for a protein, as described above) incomparison with a reference nucleic acid (e.g. a nucleic acid from theprior art) of defined length, this percentage identity is statedrelatively with reference to this reference nucleic acid. Startingtherefore, for example, from a nucleic acid which has a sequenceidentity of 50% to a reference nucleic acid 100 nucleotides long, thisnucleic acid can be a nucleic acid 50 nucleotides long which iscompletely identical to a 50 nucleotides long section of the referencenucleic acid. Indeed, it can also be a nucleic acid 100 nucleotides longwhich has 50% identity, i.e. in this case 50% identical nucleic acids,with the reference nucleic acid over the entire length thereof.Alternatively, this nucleic acid can be a nucleic acid 200 nucleotideslong which is completely identical in a 100 nucleotides long section ofthe nucleic acid to the reference nucleic acid 100 nucleotides long.Other nucleic acids of course equally meet these criteria. The identitystatements described for nucleic acids apply equally to the antibodiesand antibody fragments coded by the RNA according to the invention. Thesame holds for the determination of the sequence identity between two(poly)peptides, based on the comparison/alignment of the respectiveamino acid sequences.

The percentage identity of two sequences can be determined with the aidof a mathematical algorithm. A preferred, but not limiting, example of amathematical algorithm which can be used for comparison of two sequencesis the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877. Suchan algorithm is integrated in the NBLAST program, with which sequenceswhich have a desired identity to the sequences of the present inventioncan be identified. In order to obtain a gapped alignment, as describedhere, the “Gapped BLAST” program can be used, as is described inAltschul et al. (1997), Nucleic Acids Res, 25:3389-3402. If BLAST andGapped BLAST programs are used, the preset parameters of the particularprogram (e.g. NBLAST) can be used. The sequences can be aligned furtherusing version 9 of GAP (global alignment program) of the “GeneticComputing Group” using the preset (BLOSUM62) matrix (values −4 to +11)with a gap open penalty of −12 (for the first zero of a gap) and a gapextension penalty of −4 (for each additional successive zero in thegap). After the alignment, the percentage identity is calculated byexpressing the number of agreements as a percentage content of thenucleic acids in the sequence claimed. The methods described fordetermination of the percentage identity of two nucleic acid sequencescan also be used correspondingly, if necessary, on the coded amino acidsequences, e.g. the antibodies described here.

According to a preferred embodiment, the antibody-coding RNA accordingto the invention contains acoding regions which codes for one of theantibodies listed in Table 3. The antibody encoding RNA may be used totreat (or, may be used to provide a pharmaceutical composition to treat)one of the diseases, disorders, pathologies listed in the right-handcolumn of Table 3.

TABLE 3 Name Target Clinical application Oregovomab (OvaRex) CA125(MUC-16) Ovarian Cancer, Fallopian Tube Cancer, Peritoneal Cavity CancerCantuzumab CanAg (MUC-1) Colon Cancer, Gastric Cancer, PancreaticCancer, NSCLC HuC242-DM4 CanAg (MUC-1) Colon Cancer, Gastric Cancer,Pancreatic Cancer PAM4 (IMMU-107) CanAg (MUC-1) Pancreatic CancerHuC242-DM4 CanAg (MUC-1) Colorectal Cancer; Pancreatic Cancer HuHMFG1CanAg (MUC-1) Breast Cancer WX-G250 (Rencarex) Carbonische Renal CellCarcinoma Anhydrase IX (G250) MT103 CD19 Non-Hodgkin-LymphomaIbritumomab (Zevalin) CD20 Non-Hodgkin-Lymphoma, Lymphoma Rituximab(Rituxan, CD20 Non-Hodgkin-Lymphoma, Lymphoma, Chronic MabThera)Lymphocyte Leukemia Tositumomab (Bexxar) CD20 Non-Hodgkin-Lymphoma,Lymphoma, Myeloma Ofatumamab (HuMax-CD20) CD20 Lymphoma, B-Cell ChronicLymphocytic Leukemia Epratuzumab (LymphoCide) CD22 Non-Hodgkin-Lymphoma,Leukemia MDX-060 CD30 Hodgkin-Lymphoma, Lymphoma SGN-30 CD30Hodgkin-Lymphoma, Lymphoma Gemtuzumab (Mylotarg) CD33 LeukemiaZanolimumab (HuMax-CD4) CD4 T-Cell-Lymphoma SGN-40 CD40Non-Hodgkin-Lymphoma, Myeloma, Leukemia, Chronic Lymphocytic LeukemiaAlemtuzumab (MabCampath) CD52 T-Cell-Lymphoma, Leukemia HuN901-DM1 CD56Myeloma Galiximab CD80 Non-Hodgkin-Lymphoma Labetuzumab CEA ColonCancer, Pancreatic Cancer, Ovarian Cancer Ipilimumab (MDX-010) CTLA4Sarcoma, Melanoma, Lung cancer, Ovarian Cancer leucemia, Lymphoma, Brainand Central Nervous System Tumors, Testicular Cancer, Prostate Cancer,Pancreatic Cancer, Breast Cancer Cetuximab (Erbitux) EGFR Colon Cancer,Head and Neck Cancer, Pancreatic Cancer, Non-Small Cell Lung Cancer,Cervical Cancer, Endometrial Cancer, Breast Cancer, Myeloma, LungCancer, Gastric Cancer, Esophageal Cancer, Pancreatic Cancer,Oropharyngeal Neoplasms, Hepatocellular Carcinoma, Squamous CellCarcinoma, Sarcoma, Larynx Cancer; Hypopharynx Cancer Panitumumab(Vectibix) EGFR Colon Cancer, Lung Cancer, Breast Cancer; BladderCancer; Ovarian Cancer Nimotuzumab (TheraCim) EGFR Solid Tumors, LungCancer Matuzumab EGFR Lung Cancer, Cervical Cancer, Esophageal CancerZalutumumab EGFR Head and Neck Cancer, Squamous Cell Cancer Pertuzumab(Omnitarg) EGFR und Breast Cancer, Ovarian Cancer, Lung Cancer, HER2/neuProstate Cancer Catumaxomab (Removab) EpCam Ovarian Cancer, FallopianTube Neoplasms, Peritoneal Neoplasms MORab-003 GP-3 Ovarian Cancer,Fallopian Tube Cancer, Peritoneal Cancer MORab-009 GP-9 PancreaticCancer, Mesothelioma, Ovarian Cancer, Non-Small Cell Lung Cancer,Fallopian Tube Cancer, Peritoneal Cavity Cancer Ertumaxomab HER2/neuBreast Cancer Trastuzumab (Herceptin) HER2/neu Breast Cancer,Endometrial Cancer, Solid Tumors AMG 102 HGF Advanced Renal CellCarcinoma Apolizumab (Remitogen) HLA-DR-Antigen Solid Tumors, Leukemia,Non-Hodgkin-Lymphoma, Lymphoma CNTO 95 Integrin- Melanoma RezeptorID09C3 MHCII Non-Hodgkin-Lymphoma Denosumab (AMG-102) RANKL Myeloma,Giant Cell Tumor of Bone, Breast Cancer, Prostate Cancer GC1008 TGFbetaAdvanced Renal Cell Carcinoma; Malignant Melanoma Mapatumumab TRAIL-R1Colon Cancer, Myeloma Bevacizumab (Avastin) VEGF Colon Cancer, BreastCancer, Brain and Central Nervous System Tumors, Lung Cancer,Hepatocellular Carcinoma, Kidney Cancer, Breast Cancer, PancreaticCancer, Bladder Cancer, Sarcoma, Melanoma, Esophageal Cancer; StomachCancer, Metastatic Renal Cell Carcinoma; Kidney Cancer, Glioblastoma,Liver Cancer MEDI 522 VLA3 Solid Tumors, Leukemia, Lymphoma, Small(alpha5beta3- Intestine Cancer, Melanoma Integrin) Volociximab VLA5Renal Cell Carcinoma, Pancreatic Cancer, (alpha5beta1- MelanomaIntegrin) Name Target Application Hematology: Eculizumab (Alexion) C5Komple- Paroxysmale nächtliche Hämoglo- mentfaktor binurie (PNH)Mepolizumab Interleukin-5 Hypereosinophilie-Syndrom Dentology: CaroRx(CaroRx) Streptococcus Zahnkaries mutans Autoimmune Diseases undallergic Diseases: Efalizumab CD11a Psoriasis (Schuppenflechte)(Raptiva) Epratuzumab CD22 Autoimmune Diseases, Non-Hodgkin-(LymphoCide) Lymphom Lumiliximab CD23 Allergies Daclizumab CD25Schubförmige Multiple Sclerosis Natalizumab CD49d Multiple Sclerosis(Tysabri) Omalizumab IgE Schweres Asthma bronchiale (Xolair) (Fc-Teil)Mepolizumab Interleukin- Asthma, Hypereosinophilic Syndrome, 5Eosinophilic Gastroenteritis, Churg- Strauss Syndrome, EosinophilicEsophagitis Tocilizumab Interleukin- Rheumatoid Arthritis (Actemra) 6Adalimumab TNFα Rheumatoid Arthritis, Psoriasis- (Humira) Arthritis,Morbus Bechterew Infliximab TNFα Morbus Crohn, Rheumatoide Arthritis,(Remicade) Morbus Bechterew, Psoriasis-Arthritis, Colitis ulcerosa,Psoriasis (Schuppenflechte) Golimumab TNFα Rheumatoid Arthritis (CNTO148) Mapatumumab TRAIL-R1 Myeloma Rituximab CD20 Urticaria, RheumatoidArthritis, (Rituxan, Ulcerative Colitis, Chronic Focal MabThera)Encephalitis Epratuzumab CD22 Autoimmune diseases, Systemic Lupus(LymphoCide) Erythematosus Neurodegenerative Diseases: R1450Amyloid-beta Alzheimer Ophthalmology: Ranibizumab VEGF-A Feuchte MacularDegeneration (Lucentis) Bevacizumab VEGF Macular Degeneration (Avastin)Infektious Diseases: Palivizumab Component of RSV Prevention ofRSV-Pneumonia (Synagis) (Respiratory bei Frühgeborenen Syncytial Virus)Cardiovascular Diseases: Abciximab GPIIb/Iia Verhinderung einesGefäβverschlusses (ReoPro) nach PTCA Other Diseases: Denosumab (AMG-102)RANKL Osteoporosis GC1008 TGFbeta Pulmonary Fibrosis Bevacizumab(Avastin) VEGF Proliferative Diabetic Retinopathy

According to a preferred embodiment, the antibody-coding RNA accordingto the invention contains or has a sequence which codes for the heavychains according to SEQ ID NO: 2 and the light chains according to SEQID NO: 4. According to an even more preferred embodiment, theantibody-coding RNA according to the invention contains or has a codingsequence according to SEQ ID NO: 5 or SEQ ID NO: 51, respectively.

According to another preferred embodiment, the antibody-coding RNAaccording to the invention contains or has a sequence which codes forthe heavy chains according to SEQ ID NO: 7 and the light chainsaccording to SEQ ID NO: 9. According to an even more preferredembodiment, the antibody-coding RNA according to the invention containsor has a coding sequence according to SEQ ID NO: 10 or SEQ ID NO: 52,respectively.

According to a further preferred embodiment, the antibody-coding RNAaccording to the invention contains or has a sequence which codes forthe heavy chains according to SEQ ID NO: 12 and the light chainsaccording to SEQ ID NO: 14. According to an even more preferredembodiment, the antibody-coding RNA according to the invention containsor has a coding sequence according to SEQ ID NO: 15 or SEQ ID NO: 53,respectively.

Antibodies which are coded by the RNA according to the invention canfurthermore also encode such antibodies which have a sequence identityto one of the coding sequences of the antibodies described here, e.g. asdescribed by Table 3 or by SEQ ID NO: 5 (51), 10 (52) or 15 (53), of atleast 70%, 80% or 85%, preferably at least 90%, more preferably at least95% and most preferably at least 99% over the entire length of thenucleic acid sequence or amino acid sequence of an antibody as describedhere, e.g. as described by Table 3 or by SEQ ID NO: 5 (51), 10 (52) or15 (53).

Such antibodies which are coded by the RNA according to the inventionlikewise include antibodies according to SEQ ID NO: 5 (51), 10 (52) or15 (53) or according to Table 3 which contain or have, in one of theheavy chains described here according to SEQ ID NO: 2, 7 or 12 and/or inone of the light chains described here according to SEQ ID NO: 4, 9 or14, a nucleic acid or amino acid sequence identity of at least 70%, 80%or 85%, preferably at least 90%, more preferably at least 95% and mostpreferably at least 99% over the entire length of the coding sequencefor the particular light and/or heavy chain, with an otherwise unchangedcoding antibody sequence of SEQ ID NO: 5 (51), 10 (52) or 15 (53) or e.gantibodies of Table 3.

Overall, a novel route for carrying out antibody therapies on the basisof RNA, in particular mRNA, is thus provided with the aid of the presentinvention. In such a manner, clinically tested antibodies, for exampleangiogenesis inhibitors based on antibodies, for example bevacizumab(monoclonal immunoglobulin G₁ antibody which binds to the vasculargrowth factor VEGF (vascular endothelial growth factor); or trastuzumab(Herceptin), an indirect inhibitor which inhibits the action of tumourproteins on receptors, or for example rituximab or cetuximab (directedagainst the epidermal growth factor receptor (EGFR)), based on RNA, canbe provided, so that the inventive RNA contains at least one codingregion which codes for at least one of these antibodies.

In a preferred embodiment, the antibody-coding RNA according to theinvention typically additionally has at least one of the followingmodifications, which are preferably suitable for increasing thestability of the coding RNA, improving the expression of the antibodythereby coded, increasing the cell permeability, rendering possiblelocalization of the antibody on or in certain cell compartments etc.Each of these modifications of the RNA according to the inventiondescribed here (modified RNA) which are mentioned in the following canbe combined with one another in a suitable manner, such modificationswhich do not interfere with one another or adversely influence thestability or cell permeability of the antibody-coding, modified RNAaccording to the invention or the expression of the antibody therebycoded preferably being combined with one another. For the entire presentinvention, the nomenclature “modified” is equated with the content of“optionally modified”.

Modifications of the RNA according to the invention described here(modified RNA) can include, for example, modifications of thenucleotides of the RNA. An RNA (modified RNA) according to the inventioncan thus include, for example, backbone modifications, sugarmodifications or base modifications. In this context, theantibody-coding RNA according to the invention typically first containsnucleotides which can be chosen from all naturally occurring nucleotidesand analogues thereof (modified nucleotides), such as e.g.ribonucleotides and/or deoxyribonucleotides. Nucleotides in the contextof the present invention therefore include, without being limitedthereto, for example purines (adenine (A), guanine (G)) or pyrimidines(thymine (T), cytosine (C), uracil (U)), and as modified nucleotidesanalogues or derivatives of purines and pyrimidines, such as e.g.1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N6-isopentenyl-adenine,N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine,3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine,2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine,2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine,pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil,5-carboxymethylaminomethyl-2-thio-uracil,5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil,5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil,5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester,5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil,5′-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyaceticacid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil,queosine, β-D-mannosyl-queosine, wybutoxosine, and phosphoramidates,phosphorothioates, peptide nucleotides, methylphosphonates,7-deazaguanosine, 5-methylcytosine and inosine. The preparation of suchanalogues is known to a person skilled in the art e.g. from the U.S.Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732,U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No.4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat.No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. Nos. 5,262,530 and5,700,642, the disclosure of which is included here in its full scope byreference.

In particular, an antibody-coding RNA according to the invention cancontain RNA backbone modifications. In connection with the presentinvention, a backbone modification is a modification in which thephosphates of the backbone of the nucleotides contained in the RNA aremodified chemically. In this context, such backbone modificationstypically include, without being limited thereto, modifications from thegroup consisting of methylphosphonates, methylphosphoramidates,phosphoramidates, phosphorothioates (e.g. cytidine5′-O-(1-thiophosphate)), boranophosphates, positively chargedguanidinium groups etc., which means by replacing the phosphodiesterlinkage by other anionic, cationic or neutral groups.

An antibody-coding RNA according to the invention can likewise alsocontain sugar modifications. A sugar modification in connection with thepresent invention is a chemical modification of the sugar of thenucleotides it contains and typically includes, without being limitedthereto, sugar modifications chosen from the group consisting of2′-deoxy-2′-fluoro-oligoribonucleotide (2′-fluoro-2′-deoxycytidine5′-triphosphate, 2′-fluoro-2′-deoxyuridine 5′-triphosphate),2′-deoxy-2′-deamine-oligoribonucleotide (2′-amino-2′-deoxycytidine5′-triphosphate, 2′-amino-2′-deoxyuridine 5′-triphosphate),2′-O-alkyloligoribonucleotide, 2′-deoxy-2′-C-alkyloligoribonucleotide(2′-O-methylcytidine 5′-triphosphate, 2′-methyluridine 5′-triphosphate),2′-C-alkyloligoribonucleotide, and isomers thereof (2′-aracytidine5′-triphosphate, 2′-arauridine 5′-triphosphate), or azidotriphosphates(2′-azido-2′-deoxycytidine 5′-triphosphate, 2′-azido-2′-deoxyuridine5′-triphosphate).

Preferably, however, the modified RNA sequence according to theinvention contains no sugar modifications or backbone modifications ife.g. an in vitro transcription is necessary. The reason for thispreferred exclusion lies in the problem that certain backbonemodifications and sugar modifications of RNA sequences on the one handcan prevent or at least greatly reduce in vitro transcription thereof.Thus, an in vitro transcription of eGFP carried out by way of examplefunctions, for example, only with the sugar modifications2′-amino-2′-deoxyuridine 5′-phosphate, 2′-fluoro-2′-deoxyuridine5′-phosphate and 2′-azido-2′-deoxyuridine 5′-phosphate. In addition, thetranslation of the protein, i.e. the protein expression, in vitro or invivo typically can be reduced considerably by backbone modificationsand, independently thereof, by sugar modifications of RNA sequences. Itwas possible to demonstrate this, for example, for eGFP in connectionwith the backbone modifications and sugar modifications selected above.

An antibody-coding RNA according to the invention can likewise alsocontain modifications of the bases of the nucleotides it contains (basemodifications). Thus, for example, the antibody-coding RNA according tothe invention can be modified such that only one or several of thenucleotides of the modified RNA are exchanged for nucleotides havingbase modifications, which are preferably suitable for increasing theexpression of the antibody coded by the RNA significantly compared withthe non-modified, i.e. native RNA sequence. In this case, significantmeans an increase in the expression of the antibody on the basis of themodified RNA sequence compared with the native RNA sequence by at least20%, preferably at least 30%, 40%, 50% or 60%, even more preferably byat least 70%, 80%, 90% or even 100% and most preferably by at least150%, 200% or even 300%. In connection with the present invention, amodified nucleotide which contains a base modification is called abase-modified nucleotide and, without being limited thereto, ispreferably chosen from the group consisting of: 2-amino-6-chloropurineriboside 5′-triphosphate, 2-aminoadenosine 5′-triphosphate,2-thiocytidine 5′-triphosphate, 2-thiouridine 5′-triphosphate,4-thiouridine 5′-triphosphate, 5-aminoallylcytidine 5′-triphosphate,5-aminoallyluridine 5′-triphosphate, 5-bromocytidine 5′-triphosphate,5-bromouridine 5′-triphosphate, 5-iodocytidine 5′-triphosphate,5-iodouridine 5′-triphosphate, 5-methylcytidine 5′-triphosphate,5-methyluridine 5′-triphosphate, 6-azacytidine 5′-triphosphate,6-azauridine 5′-triphosphate, 6-chloropurine riboside 5′-triphosphate,7-deazaadenosine 5′-triphosphate, 7-deazaguanosine 5′-triphosphate,8-azaadenosine 5′-triphosphate, 8-azidoadenosine 5′-triphosphate,benzimidazole riboside 5′-triphosphate, N1-methyladenosine5′-triphosphate, N1-methylguanosine 5′-triphosphate, N6-methyladenosine5′-triphosphate, 06-methylguanosine 5′-triphosphate, pseudouridine5′-triphosphate, puromycin 5′-triphosphate or xanthosine5′-triphosphate. Nucleotides for base modifications are particularlypreferably chosen from the group of base-modified nucleotides consistingof 5-methylcytidine 5′-triphosphate and pseudouridine 5′-triphosphate.

Without being restricted thereto, in this connections the inventorsattribute an increase in the expression of the antibody coded by the(base)-modified RNA according to the invention to, inter alfa, theimprovement in the stabilizing of secondary structures and, whereappropriate, to the “more rigid” structure formed in the RNA and theincreased base stacking. Thus, for example, it is known of pseudouridine5′-triphosphate that this occurs naturally in structural RNAs (tRNA,rRNA and snRNA) in eukaryotes as well as in prokaryotes. In thisconnection, it is assumed that pseudouridine is necessary in rRNA forstabilizing secondary structures. In the course of evolution, thecontent of pseudouridine in RNA has increased, and it has been possibleto demonstrate, surprisingly, that the translation depends on thepresence of pseudouridine in the tRNA and rRNA, the interaction betweentRNA and mRNA presumably being intensified in this context. Theconversion of uridine into pseudouridine takes placeposttranscriptionally by pseudouridine synthase. A posttranscriptionalmodification of RNA likewise takes place in the case of 5-methylcytidine5′-triphosphate, and is catalysed by methyltransferases. A furtherincrease in the content of pseudouridine and the base modification ofother nucleotides is assumed to lead to similar effects, which, incontrast to the naturally occurring increased contents of pseudouridinein the sequence, can be carried out in a targeted manner and with aconsiderably wider variability. For 5-methylcytidine 5′-triphosphate andthe further base modifications mentioned here, a similar mechanism tothat for pseudouridine 5′-triphosphate is therefore assumed, i.e. animproved stabilizing of secondary structures, and on the basis of thisan improved translation efficiency. In addition to this structurallybased increase in expression, however, a positive effect on thetranslation is presumed, independently of the stabilizing of secondarystructures and a “more rigid” structure of the RNA. Further causes ofthe increase in expression are also to be found, possibly, in the lowerdegradation rate of the RNA sequences by RNAses in vitro or in vivo.

The modifications of the antibody-coding RNA according to the inventionwhich are described above can be introduced into the RNA with the aid ofmethods known to a person skilled in the art. Possible methods for thisare, for example, synthesis methods using (automatic or semiautomatic)oligonucleotide synthesis apparatuses, biochemical methods, such as e.g.in vitro transcription methods, etc. Preferably, in this connection, for(shorter) sequences which in general do not exceed a length of 50-100nucleotides, synthesis methods using (automatic or semiautomatic)oligonucleotide synthesis apparatuses and also in vitro transcriptionmethods can be employed. For (longer) sequences, e.g. sequences whichhave a length of more than 50 to 100 nucleotides, biochemical methodsare preferred, such as, for example, in vitro transcription methods,preferably an in vitro transcription method as described here,optionally using the modified RNA according to the invention.

Modifications with nucleotides as described here in an antibody-codingRNA according to the invention can occur on at least one (modifiable)nucleotide of the RNA sequence according to the invention, preferably onat least 2, 3, 4, 5, 6, 7, 8, 9 or 10 (modifiable) nucleotides, morepreferably on at least 10-20 (modifiable) nucleotides, even morepreferably on at least 10-100 (modifiable) nucleotides and mostpreferably on at least 10-200, 10 to 1,000 or 10 to 10,000 or more(modifiable), e.g. all, nucleotides. Worded alternatively, modificationsin an antibody-coding RNA according to the invention can occur on atleast one (modifiable) nucleotide of the RNA sequence according to theinvention, preferably on at least 10% of all the (modifiable)nucleotides, more preferably on at least 25% of all the (modifiable)nucleotides, even more preferably on at least 50% of all the(modifiable) nucleotides, even more preferably on at least 75% of allthe (modifiable) nucleotides and most preferably on 100% of the(modifiable) nucleotides contained in the RNA sequence according to theinvention. In this connection, a “modifiable nucleotide” is any(preferably naturally occurring (native) and therefore non-modified)nucleotide which is to be exchanged for a nucleotide modified asdescribed here. In this context, all the nucleotides of the RNA sequencecan be modified, or only certain selected nucleotides of the RNAsequence. If all the nucleotides of the RNA sequence are to be modified,100% of the “modifiable nucleotides” of the RNA sequence are all thenucleotides of the RNA sequence used. On the other hand, if only certainselected nucleotides of the RNA sequence are to be modified, theselected nucleotides are, for example, adenosine, cytidine, guanosine oruridine. Thus, for example, an adenosine of the native sequence can beexchanged for a modified adenosine, a cytidine for a modified cytidine,a uridine for a modified uridine and a guanosine for a modifiedguanosine. In this case, 100% of the “modifiable nucleotides” of the RNAsequence are 100% of the adenosines, cytidines, guanosines and/oruridines in the RNA sequence used.

According to another very preferred embodiment of the present invention,the antibody-coding RNA according to the invention can contain, forexample, a GC content which has been modified compared with the native,i.e. non-modified (precursor) RNA sequence. According to a firstalternative of the antibody-coding RNA according to the invention, theG/C content for the coding region of the RNA according to the inventionis greater than the G/C content for the coding region of the native RNAsequence, the coded amino acid sequence of the antibody or antibodyfragment being unchanged compared with the wild-type, i.e. the antibodyor antibody fragment amino acid sequence coded by the native RNAsequence. In this context, the composition and the sequence of thevarious nucleotides plays a major role. In particular, sequences havingan increased G (guanine)/C (cytosine) content are more stable thansequences having an increased A (adenine)/U (uracil) content. Accordingto the invention, the codons are therefore varied compared with thewild-type RNA, while retaining the translated amino acid sequence, suchthat they include an increased amount of G/C nucleotides. Since severalcodons code for one and the same amino acid (degeneration of the geneticcode), the most favourable codons for the stability can be determined(alternative codon usage).

Depending on the amino acid to be coded by the antibody-coding RNAaccording to the invention, there are various possibilities formodification of the native sequence of the RNA according to theinvention. In the case of amino acids which are coded by codons whichcontain exclusively G or C nucleotides, no modification of the codon isnecessary. Thus, the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala(GCC or GCG) and Gly (GGC or GGG) require no modification, since no A orU is present.

In the following cases, the codons which contain A and/or U nucleotidesare modified by substitution of other codons which encode the same aminoacids but contain no A and/or U. Examples are:

-   -   the codons for Pro can be modified from CCU or CCA to CCC or        CCG;    -   the codons for Arg can be modified from CGU or CGA or AGA or AGG        to CGC or CGG;    -   the codons for Ala can be modified from GCU or GCA to GCC or        GCG;    -   the codons for Gly can be modified from GGU or GGA to GGC or        GGG.

In other cases, although A or U nucleotides cannot be eliminated fromthe codons, it is however possible to decrease the A and U content byusing codons which contain fewer A and/or U nucleotides. For example:

-   -   the codons for Phe can be modified from UUU to UUC;    -   the codons for Leu can be modified from UUA, CUU or CUA to CUC        or CUG;    -   the codons for Ser can be modified from UCU or UCA or AGU to        UCC, UCG or AGC;    -   the codon for Tyr can be modified from UAU to UAC;    -   the stop codon UAA can be modified to UAG or UGA;    -   the codon for Cys can be modified from UGU to UGC;    -   the codon for His can be modified from CAU to CAC;    -   the codon for Gln can be modified from CAA to CAG;    -   the codons for Ile can be modified from AUU or AUA to AUC;    -   the codons for Thr can be modified from ACU or ACA to ACC or        ACG;    -   the codon for Asn can be modified from AAU to AAC;    -   the codon for Lys can be modified from AAA to AAG;    -   the codons for Val can be modified from GUU or GUA to GUC or        GUG;    -   the codon for Asp can be modified from GAU to GAC;    -   the codon for Glu can be modified from GAA to GAG.

In the case of the codons for Met (AUG) and Trp (UGG), on the otherhand, there is no possibility of sequence modification.

The substitutions listed above can of course be used individually oralso in all possible combinations to increase the G/C content of theantibody-coding RNA according to the invention compared with the nativeRNA sequence (and nucleic acid sequence, respectively). Thus, forexample, all the codons for Thr occurring in the native RNA sequence canbe modified to ACC (or ACG). Preferably, however, combinations of theabove substitution possibilities are used, e.g.:

-   -   substitution of all codons coding for Thr in the native RNA        sequence by ACC (or ACG) and substitution of all codons        originally coding for Ser by UCC (or UCG or AGC);    -   substitution of all codons coding for Ile in the native RNA        sequence by AUC and substitution of all codons originally coding        for Lys by AAG and substitution of all codons originally coding        for Tyr by UAC;    -   substitution of all codons coding for Val in the native RNA        sequence by GUC (or GUG) and substitution of all codons        originally coding for Glu by GAG and substitution of all codons        originally coding for Ala by GCC (or GCG) and substitution of        all codons originally coding for Arg by CGC (or CGG);    -   substitution of all codons coding for Val in the native RNA        sequence by GUC (or GUG) and substitution of all codons        originally coding for Glu by GAG and substitution of all codons        originally coding for Ala by GCC (or GCG) and substitution of        all codons originally coding for Gly by GGC (or GGG) and        substitution of all codons originally coding for Asn by AAC;    -   substitution of all codons coding for Val in the native RNA        sequence by GUC (or GUG) and substitution of all codons        originally coding for Phe by UUC and substitution of all codons        originally coding for Cys by UGC and substitution of all codons        originally coding for Leu by CUG (or CUC) and substitution of        all codons originally coding for Gln by CAG and substitution of        all codons originally coding for Pro by CCC (or CCG);    -   etc.

Preferably, the G/C content of the coding region of the antibody-codingRNA according to the invention is increased compared with the G/Ccontent of the coding region of the native RNA such that at least 5%, atleast 10%, at least 15%, at least 20%, at least 25% or more preferablyat least 30%, at least 35%, at least 40%, at least 45%, at least 50% orat least 55%, even more preferably at least 60%, at least 65%, at least70% or at least 75% and most preferably at least 80%, at least 85%, atleast 90%, at least 95% or at least 100% of the possible modifiablecodons of the coding region of the native RNA (and nucleic acid,respectively) are modified.

In this connection, it is particularly preferable to increase to themaximum the G/C content of the antibody-coding RNA according to theinvention, in particular in the coding region, compared with the nativeRNA sequence.

A second alternative of the antibody-coding RNA according to theinvention with modifications is based on the knowledge that thetranslation efficiency of the RNA is also determined by a differentfrequency in the occurrence of tRNAs in cells. Thus, if so-called “rare”codons are present in an RNA sequence to an increased extent, thecorresponding RNA is translated to a significantly poorer degree than inthe case where codons which code for relatively “frequent” tRNAs arepresent.

According to this second alternative of the antibody-coding RNAaccording to the invention, the coding region of the RNA according tothe invention is therefore modified compared with the coding region ofthe native RNA such that at least one codon of the native RNA whichcodes for a tRNA which is relatively rare in the cell is exchanged for acodon which codes for a tRNA which is relatively frequent in the celland which carries the same amino acid as the relatively rare tRNA.

By this modification, the sequence of the antibody-coding RNA accordingto the invention is modified such that codons for which frequentlyoccurring tRNAs are available are inserted. Which tRNAs occur relativelyfrequently in the cell and which, in contrast, are relatively rare isknown to a person skilled in the art; cf. e.g. Akashi, Curr. Opin.Genet. Dev. 2001, 11(6): 660-666.

According to the invention, by this modification all codons of thesequence of the antibody-coding RNA according to the invention whichcode for a tRNA which is relatively rare in the cell can be exchangedfor a codon which codes for a tRNA which is relatively frequent in thecell and which carries the same amino acid as the relatively rare tRNA.

It is particularly preferable to link the increased, in particularmaximum, sequential G/C content in the antibody-coding RNA according tothe invention with the “frequent” codons without modifying the aminoacid sequence coded by the RNA according to the invention. Thispreferred embodiment provides a particularly efficiently translated andstabilized RNA sequence according to the invention which encodes anantibody (for example for a pharmaceutical composition according to theinvention).

In the sequences of eukaryotic RNAs, there are typically destabilizingsequence elements (DSE) to which signal proteins bind and regulate theenzymatic degradation of the RNA in vivo. For further stabilization ofthe antibody-coding RNA according to the invention, one or moremodifications compared with the corresponding region of the native RNAare therefore optionally carried out in the region coding for theprotein, so that no destabilizing sequence elements are present.According to the invention, it is of course also preferable, whereappropriate, to eliminate from the RNA DSEs present in the untranslatedregions (3′ and/or 5′ UTR).

Such destabilizing sequences are, for example, AU-rich sequences(“AURES”), which occur in 3′ UTR sections of numerous unstable RNAs(Caput et al., Proc. Natl. Acad. Sci. USA 1986, 83: 1670 to 1674). Theantibody-coding RNA according to the invention is therefore preferablymodified compared with the native RNA such that this no longer containssuch destabilizing sequences. This also applies to those sequence motifswhich are recognized by possible endonucleases, for example the sequenceGAACAAG, which is contained in the 3′ UTR segment of the gene whichcodes for the transferrin receptor (Binder et al., EMBO J. 1994, 13:1969 to 1980). These sequence motifs are also preferably eliminated inthe antibody-coding RNA according to the invention.

A person skilled in the art is familiar with various methods which aresuitable in the present case for substitution of codons in RNAs, i.e.substitution of codons in the antibody-coding RNA according to theinvention. In the case of relatively short coding regions (which codefor antibodies or antibody fragments as described here), for example,the total antibody-coding RNA according to the invention can besynthesized chemically using standard techniques such as are familiar toa person skilled in the art.

Nevertheless, base substitutions are preferably introduced using a DNAtemplate for the preparation of the antibody-coding RNA according to theinvention with the aid of techniques of the usual targeted mutagenesis(see, for example, Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, 3rd ed., Cold SpringHarbor, N. Y., 2001). In this method, for the preparation of theantibody-coding RNA according to the invention, a corresponding DNAmolecule is therefore transcribed in vitro (see below). This DNAtemplate optionally has a suitable promoter, for example a T3, T7 or SP6promoter, for the in vitro transcription, which is followed by thedesired nucleotide sequence for the antibody-coding RNA according to theinvention to be prepared and a termination signal for the in vitrotranscription. The DNA molecule which forms the template of theantibody-coding RNA construct to be prepared can be prepared byfermentative proliferation and subsequent isolation as part of a plasmidwhich can be replicated in bacteria. Plasmids which may be mentioned assuitable for this are, for example, the plasmids pT7 Ts (GenBankaccession number U26404; Lai et al., Development 1995, 121: 2349 to2360), pGEM® series, for example pGEM®-1 (GenBank accession numberX65300; from Promega) and pSP64 (GenBank accession number X65327); cf.also Mezei and Storts, Purification of PCR Products, in: Griffin andGriffin (ed.), PCR Technology: Current Innovation, CRC Press, BocaRaton, Fla., 2001.

Using short synthetic RNA or DNA oligonucleotides which contain shortsingle-stranded transitions at the cleavage sites formed, or genesprepared by chemical synthesis, the desired nucleotide sequence can thusbe cloned into a suitable plasmid by molecular biology methods withwhich a person skilled in the art is familiar (cf. Maniatis et al.,(2001) supra). The RNA or DNA molecule is then cut out of the plasmid,in which it can be present in one or several copies, by digestion withrestriction endonucleases.

According to a particular embodiment of the present invention, theantibody-coding (modified) RNA according to the invention describedabove, especially if the RNA is in the form of mRNA, can moreover have a5′ cap structure (a modified guanosine nucleotide). Examples of capstructures which may be mentioned, without being restricted thereto, arem7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.

According to a further preferred embodiment of the present invention,the antibody-coding (modified) RNA according to the invention contains,especially if the RNA is in the form of mRNA, a poly-A tail on the 3′terminus of typically about 10 to 200 adenosine nucleotides, preferablyabout 10 to 100 adenosine nucleotides, more preferably about 20 to 70adenosine nucleotides or even more preferably about 20 to 60 adenosinenucleotides.

According to another preferred embodiment of the present invention, theantibody-coding (modified) RNA according to the invention contains,especially if the RNA is in the form of mRNA, a poly-C tail on the 3′terminus of typically about 10 to 200 cytosine nucleotides (SEQ ID NO:54), preferably about 10 to 100 cytosine nucleotides (SEQ ID NO: 55),more preferably about 20 to 70 cytosine nucleotides (SEQ ID NO: 56) oreven more preferably about 20 to 60 (SEQ ID NO: 57) or even 10 to 40cytosine nucleotides (SEQ ID NO: 58). The poly-C tail may be added tothe poly-A tail or may substitute the poly-A tail.

According to a further embodiment, the antibody-coding (modified) RNAaccording to the invention can additionally contain a nucleic acidsection which codes a tag for purification. Such tags include, butwithout being limited thereto, e.g. a hexahistidine tag (SEQ ID NO: 59)(His tag, polyhistidine tag), a streptavidin tag (Strep tag), an SBP tag(streptavidin-binding tag) a GST (glutathione S transferase) tag etc.The antibody-coding (modified) RNA according to the invention canfurthermore encode a tag for purification via an antibody epitope(antibody-binding tag), e.g. a Myc tag, an Swa11 epitope, a FLAG tag, anHA tag etc., i.e. via recognition of the epitope via the (immobilized)antibody.

For efficient translation of RNA, in particular mRNA, effective bindingof the ribosomes to the ribosome binding site (Kozak sequence:GCCGCCACCAUGG (SEQ ID NO: 16), the AUG forms the start codon) isnecessary. In this respect, it has been found that an increased A/Ucontent around this site renders possible a more efficient ribosomebinding to the RNA. According to another preferred embodiment of thepresent invention, the antibody-coding (modified) RNA according to theinvention can therefore have an increased A/U content around theribosome binding site, preferably an A/U content which is increased by 5to 50%, more preferably one increased by 25 to 50% or more, comparedwith the native RNA.

According to one embodiment of the antibody-coding (modified) RNAaccording to the invention, it is furthermore possible to insert one ormore so-called IRES (internal ribosomal entry site) into the RNA. AnIRES can thus function as the sole ribosome binding site, but it canalso serve to provide an antibody-coding (modified) RNA according to theinvention which codes for several antibodies or antibody fragments orfor at least one antibody or antibody fragment which are to betranslated by the ribosomes independently of one another(“multicistronic RNA”). Such an RNA can code, for example, a completesequence of an antibody, the corresponding coding regions of the heavyand light chain being linked (functionally) with one another by an IRESsequence. However, the heavy and light chain to be encoded by theinventive RNA may also be located in one single “cistron”. According tothe invention, the IRES sequences described are employed in particularfor (virtually) simultaneous and uniform expression of the light and theheavy chains of the antibody coded by the RNA according to theinvention. Examples of IRES sequences which can be used according to theinvention are those from picornaviruses (e.g. FMDV), pestiviruses(CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot andmouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swinefever viruses (CSFV), murine leukoma virus (MLV), simianimmunodeficiency viruses (SIV), cricket paralysis viruses (CrPV) or anSIRES sequence.

According to a further preferred embodiment of the present invention,the antibody-coding (modified) RNA according to the invention has, inthe 5′ and/or 3′ untranslated regions, stabilizing sequences which arecapable of increasing the half-life of the RNA in the cytosol. Thesestabilizing sequences can have a 100% sequence homology to naturallyoccurring sequences which occur in viruses, bacteria and eukaryotes, butcan also be partly or completely synthetic in nature. The untranslatedsequences (UTR) of the β-globin gene, for example from Homo sapiens orXenopus laevis, may be mentioned as an example of stabilizing sequenceswhich can be used in the present invention. Another example of astabilizing sequence has the general formula(C/U)CCAN_(x)CCC(U/A)Py_(x)UC(C/U)CC (SEQ ID NO: 17), which is containedin the 3′ UTR of the very stable RNA which codes for α-globin,α-(I)-collagen, 15-lipoxygenase or for tyrosine hydroxylase (cf. Holciket al., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414). Suchstabilizing sequences can of course be used individually or incombination with one another and also in combination with otherstabilizing sequences known to a person skilled in the art.

In a further preferred embodiment, the antibody-coding (modified) RNAaccording to the invention can encode a secretory signal peptide, inaddition to the antibodies as described here. Such signal peptides are(signal) sequences which conventionally comprise a length of from 15 to30 amino acids and are preferably localized on the N-terminus of thecoded antibody. Signal peptides typically render possible transport of aprotein or peptide fused therewith (here e.g. an antibody) to or into adefined cell compartment, preferably the cell surface, the endoplasmicreticulum or the endosomal-lysosomal compartment. Examples of signalsequences which can be used according to the invention are e.g. signalsequences of conventional and non-conventional MHC molecules, cytokines,immunoglobulins, the invariant chain, Lampl, tapasin, Erp57,calreticulin and calnexin, and all further membrane-located,endosomally-lysosomally or endoplasmic reticulum-associated proteins.The signal peptide of the human MHC class I molecule HLA-A*0201 ispreferably used.

Sequences which render possible transport of a protein or peptide fusedtherewith (here e.g. an antibody) to or into a defined cell compartment,preferably the cell surface, the nucleus, the nucleus region, the plasmamembrane, the cytosol, the endoplasmic reticulum, the organelles, themitochondria, the Golgi apparatus or the endosomal-lysosomalcompartment, also include, without being limited thereto, so-calledrouting signals, sorting signals, retention signals or salvage signalsand membrane topology-stop transfer signals (cf. Pugsley, A. P., ProteinTargeting, Academic Press, Inc. (1989)) at the level of the RNAaccording to the invention. In this connection, localization sequencesinclude nucleic acid sequences which encode e.g. signals, i.e. aminoacid sequences, such as, for example, KDEL (SEQ ID NO: 18) (Munro, etal., Cell 48:899-907 (1987)) DDEL (SEQ ID NO: 19), DEEL (SEQ ID NO: 20),QEDL (SEQ ID NO: 21) and RDEL (SEQ ID NO: 22) (Hangejorden, et al., J.Biol. Chem. 266:6015 (1991)) for the endoplasmic reticulum; PKKKRKV (SEQID NO: 23) (Lanford, et al. Cell 46:575 (1986)) PQKKIKS (SEQ ID NO: 24)(Stanton, L. W., et al., Proc. Natl. Acad. Sci USA 83:1772 (1986); QPKKP(SEQ ID NO: 25) (Harlow, et al., Mol. Cell Biol. 5:1605 1985), and RKKR(SEQ ID NO:26) for the nucleus; and RKKRRQRRRAHQ (SEQ ID NO: 27) (Seomi,et al., J. Virology 64:1803 (1990)), RQARRNRRRRWRERQR (SEQ ID NO: 28)(Kubota, et al., Biochem. and Biophy, Res.

Comm. 162:963 (1989)), and MPLTRRRPAASQALAPPTP (SEQ ID NO: 29) (Siomi,et al., Cell 55:197 (1988)) for the nucleus region; MDDQRDLISNNEQLP (SEQID NO: 30) (Bakke, et al., Cell 63:707-716 (1990)) for the endosomalcompartment (see, for example, Letourneur, et al., Cell 69:1183 (1992)for the targeting of liposomes). Myristoylation sequences canfurthermore be used in order to lead the expressed protein or peptide(here e.g. an antibody) to the plasma membrane, or to certain varioussub-cell compartments, such as the nucleus region, the organelles, themitochondria and the Golgi apparatus. Corresponding amino acid sequenceswhich are coded by a corresponding codon sequence of the RNA accordingto the invention are given below. The sequenceMLFNLRXXLNNAAFRHGHNFMVRNFRCGQPLX (SEQ ID NO: 31) can be used to lead theantibody to the mitochondrial matrix (Pugsley, supra). See Tang, et al.,J. Bio. Chem. 207:10122, in respect of the localization of proteins(antibodies) to the Golgi apparatus; for the localization of proteins tothe plasma membrane: GCVCSSNP (SEQ ID NO: 32), GQTVTTPL (SEQ ID NO: 33),GQELSQHE (SEQ ID NO: 34), GNSPSYNP (SEQ ID NO: 35), GVSGSKGQ (SEQ ID NO:36), GQTITTPL (SEQ ID NO: 37), GQTLTTPL (SEQ ID NO: 38), GQIFSRSA (SEQID NO: 39), GQIHGLSP (SEQ ID NO: 40), GARASVLS (SEQ ID NO: 41), andGCTLSAEE (SEQ ID NO: 42); to the endoplasmic reticulum GQNLSTSN (SEQ IDNO: 43); to the nucleus GAALTILV (SEQ ID NO: 44) and GAALTLLG (SEQ IDNO: 45); to the endoplasmic reticulum and to the cytoplasm GAQVSSQK (SEQID NO: 46) and GAQLSRNT (SEQ ID NO: 47); to the Golgi apparatus, to thenucleus, to the cytoplasm and to the cytoskeleton: GNAAAAKK (SEQ ID NO:48); to the cytoplasm and to the cytoskeleton GNEASYPL (SEQ ID NO: 49);and to the plasma membrane and to the cytoskeleton GSSKSKPK (SEQ ID NO:50). Such sequences as described above are preferably used for RNAswhich code for intrabodies, i.e antibodies which are retained in thecell and are not secreted.

The modifications described here can be introduced into theantibody-coding RNA sequence according to the invention in a suitablemanner by a person skilled in the art. For example, the optimum modifiedRNA according to the invention can be determined by methods known to theperson skilled in the art, e.g. the G/C content can be adapted manuallyand/or by means of an automated method as disclosed in WO 02/098443. Inthis context, the RNA sequences can be adapted with the variousadditional optimization aims described here: On the one hand, theadaptation can be carried out with the highest possible G/C content, andon the other hand taking into the best possible account the frequency ofthe tRNAs according to codon usage. In this context, in the first stepof the method a virtual translation of any desired RNA (or DNA) sequenceis carried out in order to generate the corresponding amino acidsequence. Starting from the amino acid sequence, a virtual reversetranslation is carried out, which on the basis of the degeneratedgenetic code provides selection possibilities for the correspondingcodons. Depending on the optimization or modification required,corresponding selection lists and optimization algorithms are used forselection of the suitable codons. The algorithm is typically implementedon a computer with the aid of suitable software. The optimized RNAsequence is established in this way and can be displayed, for example,with the aid of an appropriate display device and compared with theoriginal (wild-type) sequence. The same also applies to the frequency ofthe individual nucleotides. In this context, the changes compared withthe original nucleotide sequence are preferably highlighted. Accordingto a preferred embodiment, stable sequences which are known in natureand can provide the basis for an RNA stabilized in accordance withnatural sequence motifs are furthermore read in. A secondary structureanalysis which can analyze stabilizing and destabilizing properties or,respectively, regions of the RNA with the aid of structure calculationscan likewise be envisaged.

Furthermore, according to a preferred embodiment effective transfer ofthe antibody-coding (modified) RNA according to the invention into thecells to be treated or the organism to be treated can be improved bycomplexing the antibody-coding (modified) RNA according to the inventionwith a cationic peptide or protein or binding it thereto. Such acomplexing/condensing of the RNA, in particular mRNA, includes, forexample, complexing (or binding) of the RNA according to the inventionwith a (poly)cationic polymer, polyplexes, protein(s), in particularpolycationic protein(s), or peptide(s). Preferably, an RNA (mRNA)according to the invention is complexed or condensed with at least onecationic or polycationic agent. Preferably, such a cationic orpolycationic agent is an agent which is chosen from the group consistingof protamine, poly-L-lysine, poly-L-arginine, nucleolin, spermin andhistones, nucleolin or derivatives thereof. The use of protamine as apolycationic, nucleic acid-binding protein is particularly preferred.This procedure for stabilizing RNA is described, for example, inEP-A-1083232, the disclosure content of which in this respect isincluded in its full scope in the present invention.

According to a particular embodiment, the antibody-coding (modified) RNAaccording to the invention can contain a lipid modification. Such an RNAmodified with a lipid typically comprises an antibody-coding RNA, asdefined here, according to the invention, at least one linker covalentlylinked with this RNA and at least one lipid covalently linked with theparticular linker. Alternatively, the (modified) RNA according to theinvention modified with a lipid comprises (at least) one (modified) RNA,as defined here, according to the invention and at least one(bifunctional) lipid covalently linked with this RNA. According to athird alternative the (modified) RNA according to the invention modifiedwith a lipid comprises a (modified) RNA, as defined here, according tothe invention, at least one linker covalently linked with this RNA andat least one lipid linked covalently with the particular linker and atleast one (bifunctional) lipid covalently linked (without a linker) withthis (modified) RNA according to the invention.

The lipid employed for lipid modification of the antibody-coding(modified) RNA according to the invention is typically a lipid or alipophilic residue, which is preferably biologically active per se. Suchlipids preferably include natural substances, or compounds, such as e.g.vitamins, e.g. α-tocopherol (vitamin E), including RRR-α-tocopherol(formerly D-α-tocopherol), L-α-tocopherol, the racemateD,L-α-tocopherol, vitamin E succinate (VES) or vitamin A and derivativesthereof, e.g. retinic acid, retinol, vitamin D and derivatives thereof,e.g. vitamin D and ergosterol precursors thereof, vitamin E andderivatives thereof, vitamin K and derivatives thereof, e.g. vitamin Kand related quinone or phytol compounds, or steroids, such as bileacids, for example cholic acid, deoxycholic acid, dehydrocholic acid,cortisone, digoxygenin, testosterone, cholesterol or thiocholesterol.Further lipids or lipophilic residues in the context of the presentinvention include, without being limited thereto, polyalkylene glycols,(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), aliphatic groups,such as e.g. C₁-C₂₀-alkanes, C₁-C₂₀-alkenes, or C₁-C₂₀-alkanol compoundsetc., such as, for example, dodecanediol, hexadecanol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10, 111; Kabanov etal., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75,49), phospholipids, such as e.g. phosphatidylglycerol,diacylphosphatidylglycerol, phosphatidylcholine,dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, dihexadecyl-racglycerol,sphingolipids, cerebrosides, gangliosides, or triethylammonium1,2-di-O-hexadecylrac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), polyamines or polyalkylene glycols, such as e.g. polyethyleneglycol (PEG) (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969), hexaethylene glycol (HEG), palmitin, or palmityl residues (Mishraet al., Biochim. Biophys. Acta, 1995, 1264, 229), octadecylamines, orhexylaminocarbonyloxycholesterol residues (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923), and waxes, terpenes, alicyclichydrocarbons, saturated or mono- or polyunsaturated fatty acid residuesetc.

The linking between the lipid and the antibody-coding (modified) RNAaccording to the invention can in principle take place on anynucleotide, on the base or the sugar residue of any nucleotide, on the3′ and/or 5′ end, and/or on the phosphate backbone of theantibody-coding (modified) RNA according to the invention. According tothe invention, a terminal lipid modification of the (modified) RNAaccording to the invention on the 3′ and/or 5′ end thereof isparticularly preferred. A terminal modification has several advantagesover modifications within the sequence. On the one hand, modificationswithin the sequence can influence the hybridization properties, whichmay have an adverse effect in the case of sterically demanding residues.(Sterically demanding) modifications within the sequence very often alsointerfere in translation, which can frequently lead to an interruptionin the protein synthesis. On the other hand, in the case of preparationby synthesis of a lipid-modified (modified) RNA according to theinvention which is modified exclusively terminally, synthesis of thisantibody-coding (modified) RNA according to the invention is carried outwith monomers which are commercially available in large amounts, andsynthesis protocols known in the prior art are used.

According to a first preferred embodiment, the linking takes placebetween the antibody-coding (modified) RNA according to the inventionand at least one lipid via a linker (linked covalently with the(modified) RNA). Linkers in the context of the present inventiontypically contain at least two and optionally 3, 4, 5, 6, 7, 8, 9, 10,10-20, 20-30 or more reactive groups, chosen from e.g. a hydroxyl group,an amino group, an alkoxy group etc. One reactive group preferablyserves to bond the antibody-coding (modified) RNA according to theinvention described here. This reactive group can be in a protectedform, e.g. as a DMT (dimethoxytrityl chloride) group, as an Fmoc group,as an MMT (monomethoxytrityl) group, as a TFA (trifluoroacetic acid)group etc. Sulfur groups can furthermore be protected by disulfides,e.g. alkylthiols, such as, for example, 3-thiopropanol, or withactivated components, such as 2-thiopyridine. According to theinvention, one or more further reactive groups serve for covalentbonding of one or more lipids. According to the first embodiment, anantibody-coding (modified) RNA according to the invention can thereforebond at least one lipid via the covalently bonded linker, e.g. 1, 2, 3,4, 5, 5-10, 10-20, 20-30 or more lipid(s), particularly preferably atleast 3-8 or more lipids per (modified) RNA. In this context, the bondedlipids can be bonded separately from one another at various positions ofthe antibody-coding (modified) RNA according to the invention, but canalso be in the form of a complex at one or more positions of the(modified) RNA. An additional reactive group of the linker can be usedfor direct or indirect (cleavable) bonding to a carrier material, e.g. asolid phase. Preferred linkers according to the present invention aree.g. glycol, glycerol and glycerol derivatives,2-aminobutyl-1,3-propanediol and 2-aminobutyl-1,3-propanediolderivatives/matrix, pyrrolidine linkers or pyrrolidine-containingorganic molecules (in particular for a modification on the 3′ end) etc.According to the invention, glycerol or glycerol derivatives (C₃ anchor)or a 2-aminobutyl-1,3-propanediol derivative/matrix (C₇ anchor) areparticularly preferably used as linkers. A glycerol derivative (C₃anchor) as a linker is particularly preferred if the lipid modificationcan be introduced via an ether bond. If the lipid modification is to beintroduced e.g. via an amide or an urethane bond, e.g. a2-aminobutyl-1,3-propanediol matrix (C₇ anchor) is preferred. In thisconnection, the bond formed between the linker and the antibody-coding(modified) RNA according to the invention is preferably such that it iscompatible with the conditions and chemicals of amidite chemistry, thatis to say it is preferably neither acid- nor base-labile. In particular,those bonds which are readily accessible synthetically and are nothydrolysed by the ammoniacal cleavage procedure of a nucleic acidsynthesis process are preferred. Possible bonds are in principle allappropriately suitable bonds, preferably ester bonds, amide bonds,urethane bonds and ether bonds. In addition to the good accessibility ofthe educts (few synthesis stages), the ether bond is particularlypreferred in this context because of its relatively high biologicalstability to enzymatic hydrolysis.

According to a second preferred embodiment, for the lipid modificationof the (modified) RNA according to the invention the linking of (atleast one) (modified) RNA according to the invention takes placedirectly with at least one (bifunctional) lipid as described here, i.e.without using a linker as described here. In this case, the(bifunctional) lipid according to the invention preferably contains atleast two reactive groups, or optionally 3, 4, 5, 6, 7, 8, 9, 10 or morereactive groups, a first reactive group serving for direct or indirectbonding of the lipid to a carrier material described here and at leastone further reactive group serving for bonding of the (modified) RNA.According to the second embodiment, an antibody-coding (modified) RNAaccording to the invention can therefore preferably bond at least onelipid (directly without a linker), e.g. 1, 2, 3, 4, 5, 5-10, 10-20,20-30 or more lipid(s), particularly preferably at least 3-8 or morelipids per (modified) RNA. In this context, the bonded lipids can bebonded separately from one another at various positions of theantibody-coding (modified) RNA according to the invention, but can alsobe in the form of a complex at one or more positions of the (modified)RNA. Alternatively, according to the second embodiment, at least oneantibody-coding (modified) RNA, e.g. optionally 3, 4, 5, 6, 7, 8, 9, 10,10-20, 20-30 or more (modified) RNAs, according to the invention can bebonded to a lipid as described above via reactive groups thereof. Lipidswhich can be used for this second embodiment particularly preferablyinclude such (bifunctional) lipids which render possible a coupling(preferably on their termini or optionally intramolecularly), such ase.g. polyethylene glycol (PEG) and derivatives thereof, hexaethyleneglycol (HEG) and derivatives thereof, alkanediols, aminoalkanes,thioalkanols etc. The bond between a (bifunctional) lipid and anantibody-coding (modified) RNA according to the invention as describedabove is preferably such as is described for the first preferredembodiment.

According to a third embodiment, for the lipid modification of the(modified) RNA according to the invention the linking between theantibody-coding (modified) RNA according to the invention and at leastone lipid as described here takes place via both of the abovementionedembodiments simultaneously. Thus e.g. the antibody-coding (modified) RNAaccording to the invention can be linked at one position of the RNA withat least one lipid via a linker (analogously to the 1st embodiment) andat another position of the (modified) RNA directly with at least onelipid without using a linker (analogously to the 2nd embodiment). Forexample, at least one lipid as described here can be linked covalentlywith the RNA at the 3′ end of the (modified) RNA via a linker, and alipid as described here can be linked covalently with the RNA at the 5′end of the (modified) RNA without a linker. Alternatively, at least onelipid as described here can be linked covalently with the (modified) RNAat the 5′ end of an antibody-coding (modified) RNA according to theinvention via a linker, and a lipid as described here can be linkedcovalently with the (modified) RNA at the 3′ end of the (modified) RNAwithout a linker. Covalent linkings can likewise take place not only onthe termini of the antibody-coding (modified) RNA according to theinvention, but also intramolecularly, as described above, e.g. on the 3′end and intramolecularly, on the 5′ end and intramolecularly, on the 3′and 5′ end and intramolecularly, exclusively intramolecularly etc.

The (modified) RNA according to the invention described here can beprepared by preparation processes known in the prior art, e.g.automatically or manually via known nucleic acid syntheses (see, forexample, Maniatis et al. (2001) supra) or also via molecular biologymethods, for example with subsequent purification, for example viachromatography methods.

According to further subject matter of the present invention, theantibody-coding (modified) RNA according to the invention can be usedfor the preparation of a pharmaceutical composition for treatment oftumours and cancer diseases, cardiovascular diseases, infectiousdiseases, autoimmune diseases or optionally monogenetic diseases, e.g.in gene therapy.

A pharmaceutical composition in the context of the present inventioncomprises an antibody-coding (modified) RNA as described here andoptionally a pharmaceutically suitable carrier and/or further auxiliarysubstances and additives. The pharmaceutical composition employedaccording to the present invention typically comprises a safe andeffective amount of a (modified) RNA as described here. As used here,“safe and effective amount” means an amount of the antibody-coding(modified) RNA according to the invention such as is sufficient toinduce significantly, by expression of the coded antibody, a positivechange of a state to be treated, e.g. a tumour disease or cancerdisease, a cardiovascular disease or an infectious disease, as describedin the following. At the same time, however, a “safe and effectiveamount” is low enough to avoid serious side effects in the therapy ofthese diseases, that is to say to render possible a reasonable ratio ofadvantage and risk. Determination of these limits typically lies withinthe range of reasonable medical judgement. The concentration of theantibody-coding (modified) RNA according to the invention in suchpharmaceutical compositions can therefore vary, for example, withoutbeing limited thereto, within a wide range of from e.g. 0.1 ng to 1,000mg/ml. Such a “safe and effective amount” of an antibody-coding(modified) RNA according to the invention can vary in connection withthe particular state to be treated and the age and the physical state ofthe patient to be treated, the severity of the state, the duration ofthe treatment, the nature of the concomitant therapy, of the particularpharmaceutically suitable carrier used and similar factors within theknowledge and experience of the treating doctor. The pharmaceuticalcomposition described here can be employed for human and also forveterinary medicine purposes.

The pharmaceutical composition according to the invention described herecan optionally comprise a pharmaceutically suitable carrier (and/orvehicle). The term “pharmaceutically suitable carrier (and/or vehicle)”used here preferably includes one or more compatible solid or liquidcarriers or vehicles, (e.g. fillers, or diluents or encapsulatingcompounds) which are suitable for administration to a person. The term“compatible” as used here means that the constituents of the compositionare capable of being mixed together with the antibody-coding (modified)RNA according to the invention and the auxiliary substance optionallycontained in the composition, as such and with one another in a mannersuch that no interaction occurs which would substantially reduce thepharmaceutical effectiveness of the composition under usual condition ofuse, such as e.g. would reduce the pharmaceutical activity of the codedantibody or even suppress or impair expression of the coded antibody.Pharmaceutically suitable carrier must of course have a sufficientlyhigh purity and a sufficiently low toxicity to render them suitable foradministration to a person to be treated.

Pharmaceutically suitable carriers or vehicles, that may be used in theinventive pharmaceutical composition, may be typically distinguishedinto solid or liquid carriers or vehicles, wherein a specificdetermination may depend on the viscosity of the respective carrier orvehicle to be used.

In this context, solid carriers and vehicles typically include e.g., butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, and salts, if provided in solid form, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, orpolyvinyl pyrrolidone, waxes, polyethylenepolyoxypropylene-blockpolymers, wool fat, sugars, such as, for example, lactose, glucose andsucrose; starches, such as, for example, corn starch or potato starch;or cellulose-based substances, e.g. cellulose and its derivatives, suchas, for example, sodium carboxymethylcellulose, ethylcellulose,cellulose acetate; pulverized tragacanth; malt; gelatine; tallow; solidlubricants, such as, for example, stearic acid, magnesium stearate;calcium sulfate; wetting agents, such as, for example, sodium laurylsulfate; colouring agents; flavouring agents; drug (active agent)carriers; tablet-forming agents; stabilizers; antioxidants;preservatives; etc. . . .

Liquid carriers or vehicles, e.g. for aqueous or oleaginous suspensions,typically include, but are not limited to, e.g., water; pyrogen-freewater; solutions of ion exchangers, alumina, aluminum stearate,lecithin, or serum proteins, such as human serum albumin; alginic acid;isotonic saline solutions or phosphate-buffered solutions, Ringer'ssolution, isotonic sodium chloride solution, etc. or salts orelectrolytes, if provided in solubilized form, such as protaminesulfate, phosphates, e.g. disodium hydrogen phosphate, potassiumhydrogen phosphate, sodium chloride, zinc salts, or (other) buffersubstances including e.g. glycine, sorbic acid, potassium sorbate;liquid solutions of polyols, such as, for example, polyethylene glycol,polypropylene glycol, glycerol, 1,3-butanediol, sorbitol, Mannitol;sterile, fixed oils, any suitable bland fixed oil, e.g. includingsynthetic mono- or di-glycerides, partial glyceride mixtures ofsaturated vegetable fatty acids, fatty acids, such as oleic acid and itsglyceride derivatives, natural pharmaceutically-acceptable oils, e.g.plant oils, such as, for example, groundnut oil, cottonseed oil, sesameoil, corn oil and oil from Theobroma; olive oil or castor oil,especially in their polyoxyethylated versions. These liquid carriers orvehicles may also contain or comprise a long-chain alcohol diluent ordispersant, such as carboxymethyl cellulose or similar dispersingagents, or commonly used surfactants or emulsifiers, such as Tween®,Spans and other emulsifying agents or bioavailability enhancers, etc.,if provided in a liquid form.

The choice of a pharmaceutically suitable carrier as described above isdetermined in particular by the mode in which the pharmaceuticalcomposition according to the invention is administered. Thepharmaceutical composition according to the invention can beadministered, for example, systemically. Administration routes includee.g. transdermal, oral, parenteral, including subcutaneous orintravenous injections, topical and/or intranasal routes. The suitableamount of the pharmaceutical composition according to the inventionwhich is to be used can be determined by routine experiments usinganimal models. Such models include, but without being limited thereto,models of the rabbit, sheep, mouse, rat, dog and non-human primatemodels. Preferred unit dose forms for injection include sterilesolutions of water, physiological saline solution or mixtures thereof.The pH of such solutions should be adjusted to about 7.4. Suitablecarriers for injection include hydrogels, devices for controlled ordelayed release, polylactic acid and collagen matrices. Pharmaceuticallysuitable carriers which can be used here include those which aresuitable for use in lotions, creams, gels and the like. If the compoundis to be administered perorally, tablets, capsules and the like are thepreferred unit dose form. The pharmaceutically suitable carriers for thepreparation of unit dose forms which can be used for oral administrationare wellknown in the prior art. Their choice will depend on secondaryconsiderations, such as flavour, cost and storage stability, which arenot critical for the purposes of the present invention and can beimplemented without difficulties by a person skilled in the art.

The pharmaceutical composition according to the invention canfurthermore comprise an injection buffer, which preferably improves thetransfection and also the translation of the antibody-coding RNAaccording to the invention in cells or an organism. The pharmaceuticalcomposition according to the invention can comprise, for example, anaqueous injection buffer which contains, with respect to the totalpharmaceutical composition, if this is in liquid form, a sodium salt,preferably at least 50 mM sodium salt, a calcium salt, preferably atleast 0.01 mM calcium salt, and optionally a potassium salt, preferablyat least 3 mM potassium salt. According to a preferred embodiment, thesodium salts, calcium salts and optionally potassium salts contained insuch an injection buffer are in the form of halides, e.g. chlorides,iodides or bromides, or in the form of their hydroxides, carbonates,bicarbonates or sulfates. Examples which are to be mentioned here are,for the sodium salt NaCl, NaI, NaBr, Na₂CO₃, NaHCO₃, Na₂SO₄, for thepotassium salt optionally present KCl, KI, KBr, K₂CO₃, KHCO₃, K₂SO₄, andfor the calcium salt CaCl₂, CaI₂, CaBr₂, CaCO₃, CaSO₄, Ca(OH)₂. Theinjection buffer can also contain organic anions of the abovementionedcations. In a particularly preferred embodiment, such an injectionbuffer contains as salts sodium chloride (NaCl), calcium chloride(CaCl₂) and optionally potassium chloride (KCl), it also being possiblefor other anions to be present in addition to the chlorides.

These salts are typically present in the injection buffer optionallyused in the pharmaceutical composition according to the invention, withrespect to the total pharmaceutical composition (if this is in liquidform), in a concentration of at least 50 mM sodium chloride (NaCl), atleast 3 mM potassium chloride (KCl) and at least 0.01 mM calciumchloride (CaCl₂). The injection buffer can be in the form of bothhypertonic and isotonic or hypotonic injection buffers. In connectionwith the present invention, in this context the injection buffer ishypertonic, isotonic or hypotonic with respect to the particularreference medium, i.e. the injection buffer has either a higher, thesame or a lower salt content compared with the particular referencemedium, such concentrations of the abovementioned salts which do notlead to damage to the cells caused by osmosis or other concentrationeffects preferably being employed. Reference media here are, forexample, liquids which occur in “in vivo” methods, such as, for example,blood, lymph fluid, cytosol fluids or other fluids which occur in thebody, or liquids or buffers conventionally employed in “in vitro”methods. Such liquids and buffers are known to a person skilled in theart.

The injection buffer optionally contained in the pharmaceuticalcomposition according to the invention can also contain furthercomponents, for example sugars (mono-, di-, tri- or polysaccharides), inparticular glucose or mannitol. In a preferred embodiment, however, nosugars are present in the injection buffer used. It is also preferablefor the injection buffer precisely to contain no non-charged components,such as, for example, sugars. The injection buffer typically containsexclusively metal cations, in particular from the group consisting ofthe alkali or alkaline earth metals, and anions, in particular theanions described above. The pH of the injection buffer used, withrespect to the total pharmaceutical composition, if this is in liquidform, is preferably between 1 and 8.5, preferably between 3 and 5, morepreferably between 5.5 and 7.5, in particular between 5.5 and 6.5. Ifappropriate, the injection buffer can also contain a buffer system whichfixes the injection buffer at a buffered pH. This can be, for example, aphosphate buffer system, HEPES or Na₂HPO₄/NaH₂PO₄. However, theinjection buffer used very particularly preferably contains none of theabovementioned buffer systems or contains no buffer system at all.

The injection buffer optionally contained in the pharmaceuticalcomposition according to the invention can contain, in addition to or asan alternative to the monovalent and divalent cations described,divalent cations, in particular from the group consisting of alkalineearth metals, such as, for example, magnesium (Mg²⁺), or also iron(Fe²⁺), and monovalent cations, in particular from the groups consistingof alkali metals, such as, for example, lithium (Li⁺). These monovalentcations are preferably in the form of their salts, e.g. in the form ofhalides, e.g. chlorides, iodides or bromides, or in the form of theirhydroxides, carbonates, bicarbonates or sulfates. Examples which are tobe mentioned here are, for the lithium salt LiCl, LiI, LiBr, Li₂CO₃,LiH—CO₃, Li₂SO₄, for the magnesium salt MgCl₂, MgI₂, MgBr₂, MgCO₃,MgSO₄, and Mg(OH)₂, and for the iron salt FeCl₂, FeBr₂, FeI₂, FeF₂,Fe₂O₃, FeCO₃, FeSO₄, Fe(OH)2. All the combinations of di- and/ormonovalent cations, as described above, are likewise included. Suchinjection buffers which contain only divalent, only monovalent or di-and monovalent cations can thus be used in the pharmaceuticalcomposition according to the invention. Such injection buffers whichcontain only one type of di- or monovalent cations, particularlypreferably e.g. only Ca²⁺ cations, or a salt thereof, e.g. CaCl₂, canlikewise be used. The molarities given above for Ca²⁺ (as a divalentcation) and Na¹⁺ (as a monovalent cation) (that is to say typicallyconcentrations of at least 50 mM Na⁺, at least 0.01 mM Ca²⁺ andoptionally at least 3 mM K⁺) in the injection buffer can also be takeninto consideration if another di- or monovalent cation, in particularother cations from the group consisting of the alkaline earth metals andalkali metals, are employed instead of some or all the Ca²⁺ or,respectively, Na¹⁺ in the injection buffer used according to theinvention for the preparation of the injection solution. All the Ca²⁺ orNa¹⁺ as mentioned above, can indeed be replaced by other di- or,respectively, monovalent cations in the injection buffer used, forexample also by a combination of other divalent cations (instead ofCa²⁺) and/or a combination of other monovalent cations (instead of Na¹⁺)(in particular a combination of other divalent cations from the groupconsisting of the alkaline earth metals or, respectively, of othermonovalent cations from the group consisting of the alkali metals), butit is preferable to replace at most some of the Ca²⁺ or Na¹⁺, i.e. forat least 20%, preferably at least 40%, even more preferably at least 60%and still more preferably at least 80% of the particular totalmolarities of the mono- and divalent cations in the injection buffer tobe occupied by Ca²⁺ and, respectively, Na¹⁺. However, it is veryparticularly preferable if the injection buffer optionally contained inthe pharmaceutical composition according to the invention containsexclusively Ca²⁺ as a divalent cation and Na¹⁺ as a monovalent cation,that is to say, with respect to the total pharmaceutical composition,Ca²⁺ represents 100% of the total molarity of divalent cations, just asNa¹⁺ represents 100% of the total molarity of monovalent cations. Theaqueous solution of the injection buffer can contain, with respect tothe total pharmaceutical composition, up to 30 mol % of the saltscontained in the solution, preferably up to 25 mol %, preferably up to20 mol %, furthermore preferably up to 15 mol %, more preferably up to10 mol %, even more preferably up to 5 mol %, likewise more preferablyup to 2 mol % of insoluble or sparingly soluble salts. Salts which aresparingly soluble in the context of the present invention are those ofwhich the solubility product is <10′. Salts which are readily solubleare those of which the solubility product is >10′. Preferably, theinjection buffer optionally contained in the pharmaceutical compositionaccording to the invention is from 50 mM to 800 mM, preferably from 60mM to 500 mM, more preferably from 70 mM to 250 mM, particularlypreferably 60 mM to 110 mM in sodium chloride (NaCl), from 0.01 mM to100 mM, preferably from 0.5 mM to 80 mM, more preferably from 1.5 mM to40 mM in calcium chloride (CaCl₂) and optionally from 3 mM to 500 mM,preferably from 4 mM to 300 mM, more preferably from 5 mM to 200 mM inpotassium chloride (KCl). Organic anions can also occur as furtheranions in addition to the abovementioned inorganic anions, for examplehalides, sulfates or carbonates. Among these there may be mentionedsuccinate, lactobionate, lactate, malate, maleate etc., which can alsobe present in combination.

An injection buffer optionally contained in the pharmaceuticalcomposition according to the invention preferably contains lactate. Ifit contains an organic anion, such an injection buffer particularlypreferably contains exclusively lactate as the organic anion. Lactate inthe context of the invention can be any desired lactate, for exampleL-lactate and D-lactate. Lactate salts which occur in connection withthe present invention are typically sodium lactate and/or calciumlactate, especially if the injection buffer contains only Na⁺ as amonovalent cation and Ca²⁺ as a divalent cation. An injection bufferoptionally used in the pharmaceutical composition according to theinvention and as described above preferably contains, with respect tothe total pharmaceutical composition, from 15 mM to 500 mM, morepreferably from 15 mM to 200 mM, and even more preferably from 15 mM to100 mM lactate. In this context, it has been found that the use of aninjection buffer with the components described above, optionally with orwithout lactate (in the following: “RL injection buffer” if it does notcontain the component lactate, or “RL injection buffer with lactate” ifit does contain the component lactate) for RNA injection solutions (i.e.injection solutions which contain RNA and are suitable for injection ofthis RNA) significantly increases both the transfer and the translationof the RNA into/in the cells/tissue of a host organism (mammal) comparedwith other injection buffers conventionally used in the prior art.

According to a particular embodiment, the pharmaceutical compositionused here can also be provided as a passive vaccine (for passiveimmunization). In the present invention, without being restricted to atheory, passive immunization is based on the introduction of anantibody-coding (modified) RNA as described here into an organism, inparticular into a cell, the coded antibody then being expressed, i.e.translated. As a result, binding of such molecules, e.g. nucleic acidsor antigens, for which the coded antibody is specific can take place.Passive vaccines in connection with the present invention typicallycomprise a composition as described above for a pharmaceuticalcomposition, the composition of such passive vaccines used beingdetermined in particular by the mode in which they are administered.Preferably, the passive vaccines according to the invention areadministered systemically or in some cases non-systemically.Administration routes of such passive vaccines according to theinvention typically include transdermal, oral, parenteral, includingsubcutaneous, intravenous, or intraarterial injections, topical and/orintranasal routes. Passive vaccines according to the invention aretherefore preferably formulated in a liquid or solid form.

According to further preferred subject matter of the present invention,the antibody-coding (modified) RNA according to the invention or apharmaceutical composition according to the invention is used fortreatment of indications mentioned by way of example in the following.Without being limited thereto, diseases or states, for example, such ase.g. cancer or tumour diseases chosen from melanomas, malignantmelanomas, colon carcinomas, lymphomas, sarcomas, blastomas, kidneycarcinomas, gastrointestinal tumours, gliomas, prostate tumours, bladdercancer, rectal tumours, stomach cancer, oesophageal cancer, pancreaticcancer, liver cancer, mammary carcinomas (=breast cancer), uterinecancer, cervical cancer, acute myeloid leukaemia (AML), acute lymphoidleukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocyticleukaemia (CLL), hepatomas, diverse virus-induced tumours, such as e.g.papilloma virus-induced carcinomas (e.g. cervix carcinoma=cervicalcancer), adenocarcinomas, herpes virus-induced tumours (e.g. Burkitt'slymphoma, EBV-induced B cell lymphoma), hepatitis B-induced tumours(hepatocell carcinomas), HTLV-1- and HTLV-2-induced lymphomas, acusticusneurinoma, lung carcinomas (=lung cancer=bronchial carcinoma), smallcell lung carcinomas, throat cancer, anal carcinoma, glioblastoma,rectum carcinoma, astrocytoma, brain tumours, retinoblastoma, basalioma,brain metastases, medulloblastomas, vaginal cancer, testicular cancer,thyroid carcinoma, Hodgkin's syndrome, meningeomas, Schneeberger'sdisease, pituitary tumour, mycosis fungoides, carcinoids, neurinoma,spinalioma, Burkitt's lymphoma, laryngeal cancer, kidney cancer,thymoma, corpus carcinoma, bone cancer, non-Hodgkin's lymphomas,urethral cancer, CUP syndrome, head/neck tumours, oligodendroglioma,vulval cancer, intestinal cancer, colon carcinoma, oesophageal carcinoma(=oesophageal cancer), wart conditions, small intestine tumours,craniopharyngeomas, ovarian carcinoma, soft tissue tumours (sarcomas),ovarian cancer (=ovarian carcinoma), pancreatic carcinoma (=pancreaticcancer), endometrium carcinoma, liver metastases, penis cancer, tonguecancer, gallbladder cancer, leukaemia, plasmocytoma, lid tumour,prostate cancer (=prostate tumours) etc., or infectious diseases suchas, for example, influenza, malaria, SARS, yellow fever, AIDS, Lymeborreliosis, leishmaniasis, anthrax, meningitis, can be treated with thepharmaceutical composition described.

The antibody-coding (modified) RNA according to the invention or apharmaceutical composition according to the invention can likewise beused for treatment of, for example, viral infectious diseases chosenfrom, without being limited thereto, AIDS, condyloma acuminata,molluscum contagiosum, dengue fever, three-day fever, Ebola virus,colds, early summer meningoencephalitis (ESME), influenza, shingles,hepatitis, herpes simplex type I, herpes simplex type II, herpes zoster,influenza, Japanese encephalitis, Lassa fever, Marburg virus, measles,foot and mouth disease, mononucleosis, mumps, Norwalk virus infection,Pfeiffer's glandular fever, smallpox, polio (poliomyelitis),pseuodcroup, infectious erythema, rabies, warts, West Nile fever,chicken-pox, cytomegalovirus (CMV), caused by viruses chosen from,without being limited thereto, e.g. HIV, orthopox variola virus,orthopox alastrim virus, parapox ovis virus, molluscum contagiosumvirus, herpes simplex virus 1, herpes simplex virus 2, herpes B virus,varicella zoster virus, pseudorabies virus, human cytomegaly virus,human herpes virus 6, human herpes virus 7, Epstein-Barr virus, humanherpes virus 8, hepatitis B virus, chikungunya virus, O'nyong'nyongvirus, rubivirus, hepatitis C virus, GB virus C, West Nile virus, denguevirus, yellow fever virus, louping ill virus, St. Louis encephalitisvirus, Japan B encephalitis virus, Powassan virus, FSME virus,SARS-associated corona virus, human corona virus 229E, human coronavirus Oc43, Torovirus, human T cell lymphotropic virus type I, human Tcell lymphotropic virus type II, human immunodeficiency virus type 1,human immunodeficiency virus type 2, Lassa virus, lymphocyticchoriomeningitis virus, Tacaribe virus, Junin virus, Machupo virus,Borna disease virus, Bunyamwera virus, California encephalitis virus,Rift Valley fever virus, sand fly fever virus, Toscana virus,Crimean-Congo haemorrhagic fever virus, Hazara virus, Khasan virus,Hantaan virus, Seoul virus, Prospect Hill virus, Puumala virus, DobravaBelgrade virus, Tula virus, sin nombre virus, Lake Victoria Marburgvirus, Zaire Ebola virus, Sudan Ebola virus, Ivory Coast Ebola virus,influenza virus A, influenza virus B, influenza viruses C, parainfluenzavirus, measles virus, mumps virus, respiratory syncytial virus, humanmetapneumovirus, vesicular stomatitis Indiana virus, rabies virus,Mokola virus, Duvenhage virus, European bat lyssavirus 1+2, Australianbat lyssavirus, adenoviruses A-F, human papilloma viruses, condylomavirus 6, condyloma virus 11, polyoma viruses, adeno-associated virus 2,rotaviruses, or orbiviruses etc.,

or bacterial infectious diseases, such as abortion (infectious, septic),prostatitis (prostate inflammation), anthrax, appendicitis (inflammationof the caecum), borreliosis, botulism, Campylobacter, Chlamydiatrachomatis (inflammation of the urethra, conjunctiva), cholera,diphtheria, donavonosis, epiglottitis, louse-borne typhus, typhoidfever, gas gangrene, gonorrhoea, hare plague, Helicobacter pylori,whooping-cough, climatic bubo, osteomyelitis, legionnaires' disease,leprosy, listeriosis, pneumonia, meningitis, bacterial meningitis,anthrax, inflammation of the middle ear, Mycoplasma hominis, neonatalsepsis (chorioamnionitis), noma, paratyphoid fever, plague, Reiter'ssyndrome, Rocky Mountain spotted fever, Salmonella paratyphoid fever,Salmonella typhoid fever, scarlet fever, syphilis, tetanus, gonorrhoea,tsutsugamushi fever, tuberculosis, typhus, vaginitis (colpitis), softchancre and infectious diseases caused by parasites, protozoa or fungi,such as amoebic dysentery, bilharziosis, Chagas' disease, Echinococcus,fish tapeworm, ichthyotoxism (ciguatera), fox tapeworm, mycosis pedis,dog tapeworm, candiosis, ptyriasis, the itch (scabies), cutaneousleishmaniasis, lamblian dysentery (giadiasis), lice, malaria,onchocercosis (river blindness), fungal diseases, beef tapeworm,schistosomiasis, sleeping sickness, pork tapeworm, toxoplasmosis,trichomoniasis, trypanosomiasis (sleeping sickness), visceralleishmaniasis, nappy dermatitis or dwarf tapeworm.

The antibody-coding (modified) RNA according to the invention or apharmaceutical composition according to the invention can also be usedfor treatment of cardiovascular diseases chosen from, without beinglimited thereto, coronary heart disease, arteriosclerosis, apoplexy andhypertension, and neuronal diseases chosen from Alzheimer's disease,amyotrophic lateral sclerosis, dystonia, epilepsy, multiple sclerosisand Parkinson's disease etc.

The antibody-coding (modified) RNA according to the invention or apharmaceutical composition according to the invention can furthermore beused for treatment of autoimmune diseases chosen from, without beinglimited thereto, autoimmune type I diseases or autoimmune type IIdiseases or autoimmune type III diseases or autoimmune type IV diseases,such as, for example, multiple sclerosis (MS), rheumatoid arthritis,diabetes, diabetes type I (diabetes mellitus), systemic lupuserythematosus (SLE), chronic polyarthritis, Basedow's disease,autoimmune forms of chronic hepatitis, colitis ulcerosa, allergy type Idiseases, allergy type II diseases, allergy type III diseases, allergytype IV diseases, fibromyalgia, hair loss, Bechterew's disease, Crohn'sdisease, myasthenia gravis, neurodermatitis, polymyalgia rheumatica,progressive systemic sclerosis (PSS), psoriasis, Reiter's syndrome,rheumatic arthritis, psoriasis, vasculitis etc., or diabetes type II.

Diseases in the context of the present invention likewise includemonogenetic diseases, i.e. (hereditary) diseases which are caused by anindividual gene defect and are inherited according to Mendel's rules.Monogenetic diseases in the context of the present invention arepreferably chosen from the group consisting of autosomally recessivehereditary diseases, such as, for example, adenosine deaminasedeficiency, familial hypercholesterolaemia, Canavan's syndrome,Gaucher's disease, Fanconi's anaemia, neuronal ceroid lipofuscinoses,mucoviscidosis (cystic fibrosis), sickle cell anaemia, phenylketonuria,alcaptonuria, albinism, hypothyroidism, galactosaemia, alpha-1antitrypsin deficiency, xeroderma pigmentosum, Ribbing's syndrome,mucopolysaccharidoses, cleft lip, jaw, palate,Laurence-Moon-Biedl-Bardet syndrome, short rib polydactyly syndrome,cretinism, Joubert's syndrome, progeria type II, brachydactyly,adrenogenital syndrome, and X chromosomal hereditary diseases, such as,for example, colour blindness, e.g. red-green blindness, fragile Xsyndrome, muscular dystrophy (Duchenne and Becker-Kiener type),haemophilia A and B, G6PD deficiency, Fabry's disease,mucopolysaccharidosis, Norrie's syndrome, retinitis pigmentosa, septicgranulomatosis, X-SCID, ornithine transcarbamylase deficiency,Lesch-Nyhan syndrome, or from autosomally dominant hereditary diseases,such as, for example, hereditary angioedema, Marfan's syndrome,neurofibromatosis, progeria type I, osteogenesis imperfecta,Klippel-Trenaunay syndrome, Sturge-Weber syndrome, Hippel-Lindausyndrome and tuberous sclerosis. RNA according to the invention whichencodes an antibody as described here can be used on monogeneticdiseases in the context of the present invention, the coded antibodybeing able to intervene in a regulatory manner, and also as a therapy,for example by regulation of undesirable metabolism products, trappingof specific gene products, interference with undesired certaininteractions of proteins, e.g. inhibiting certain undesiredligand/receptor interactions etc.

A (modified) RNA according to the invention which encodes an antibodycan be employed in various ways for treatment of the abovementionedindications. Thus, cancer diseases, for example, can be treated byimmunotherapy in addition or as an alternative to known therapies. Forthis, for example, an RNA according to the invention which codes for abispecific antibody can be employed, the antibody recognizing on the onehand a surface antigen, such as e.g. CD3, on T cells and on the otherhand a tumour antigen, such as e.g. Her2/neu, C20, EGFR or CA-125. As aresult, T cells which are positive in respect of certain surfaceantigens and tumour cells which express the tumour antigen are broughtspatially close, which improves the recognition of the tumour cells bythe immune system and therefore increases the destruction of the tumourcells.

Furthermore, e.g. in cardiac infarction cases, for example, it ispossible to employ an RNA according to the invention which codes for abispecific antibody which recognizes on the one hand a stem cellantigen, such as e.g. CD45, and on the other hand an antigen of thetarget tissue, such as e.g. myosin light chain, in order to increase theconcentration of stem cells in the heart muscle (see also Reusch et al.Anti-CD3 x anti-epidermal growth factor receptor (EGFR) bispecificantibody redirects T-cell cytolytic activity to EGFR-positive cancers invitro and in an animal model. Clin Cancer Res. 2006).

Furthermore, by using RNA according to the invention which codesbispecific antibodies, e.g. two different cell types can be brought intocontact or spatially close by the coded antibodies. This isadvantageous, for example, in order to concentrate a cell in a tissue orto bring two proteins, e.g. antigens, into contact with or spatiallyclose to one another, e.g. ligand and receptor or proteins which mustdimerize/oligomerize in order to become activated.

RNAs according to the invention as described here which code forintrabodies can also be employed for use on the abovementioned diseases,in particular infectious diseases, autoimmune diseases and neuronaldiseases and also on monogenetic diseases. Thus, intrabodies can be usedin order to inhibit, as e.g. bispecific intracellularly expressedantibodies, cytoplasmic proteins (be it proteins originating from thepathogenic organism or be it proteins from the host organism), asdescribed above. For example, RNAs according to the invention which codefor intrabodies can be employed in order to inhibit IL-2R (receptor ofIL-2) or ErbB2 (Her2/neu) by the coded antibodies. RNAs according to theinvention which code for intrabodies are also suitable for use on virusdiseases, such as e.g. HIV-1. It has furthermore been possible todemonstrate e.g. that infection of mice with scrapie, a prion disease,can be prevented by expression of an scFv fragment against the prionprotein (Vertrugno et al., KDEL-tagged (“KDEL” disclosed as SEQ ID NO:18) anti-prion intrabodies impair PrP lysosomal degradation and inhibitscrapie infectivity., Biochem Biophys Res Commun. 2005; Marasco Wash.,Intrabodies: turning the humoral immune system outside in forintracellular immunization, Gene Therapy (1997) 4: 11-15). RNAsaccording to the invention which code for intrabodies can furthermore beemployed in order to bind and preferably to neutralize, by the codedantibodies, intracellularly expressed factors as described here, such ase.g. antigens, nucleic acids etc. (see above).

In this connection, the invention therefore also provides the use of anantibody-coding (modified) RNA according to the invention or of apharmaceutical composition according to the invention, e.g. a passivevaccine according to the invention, for treatment of indications anddiseases described here. This also includes, in particular, the use ofthe antibody-coding (modified) RNA according to the invention forpassive immunization and, respectively, the use of the pharmaceuticalcomposition described according to the invention as a passive vaccine.The use of the antibody-coding (modified) RNA according to the inventionfor the preparation of a pharmaceutical composition or a passivevaccine, as described here, for treatment of the indications describedhere is likewise included. The use of the antibody-coding (modified) RNAaccording to the invention for the preparation of a passive vaccine, asdescribed here, for passive immunization against the abovementionedindications is also included.

In this connection, the invention therefore likewise provides the use ofan antibody-coding (modified) RNA according to the invention, of theantibody thereby coded, of the pharmaceutical composition described hereor of the passive vaccine according to the invention for therapeutic useor for inhibition/neutralization of a protein function in one of theindications described here. In this context, a protein function ispreferably suppressed (neutralizing antibodies). In principle, any ofthe antibodies coded by the RNA according to the invention and describedhere simultaneously also has a neutralizing action by binding of itsspecific substrate. Examples include e.g. anti-CD4 antibodies forprevention of rejection of transplants, Avastin (see above), Herceptin(see above) etc.

In this connection, the invention therefore also furthermore providesthe use of an antibody-coding (modified) RNA according to the invention,of the antibody thereby coded or of the pharmaceutical compositiondescribed here for therapeutic use for passive immunization bytriggering an immunological effector function in the sense of amonoclonal antibody. In this context, e.g. therapy of tumour cells orpathogens, such as viruses or bacteria, in the indications as describedhere is rendered possible by expression and secretion of the antibody orantibody fragment. Hereby, the immune defense of the host is supportedby the inventive RNA by triggering the innate, cellular or humoralimmune system. Antibodies may be directed against immune suppressingfactors or they may simulate the function of certain immunologicallyactive cytokines by e.g. activating cytokine receptors.

Furthermore, in this connection an antibody-coding (modified) RNAaccording to the invention or the pharmaceutical composition accordingto the invention described here or the passive vaccine according to theinvention can also be used as an immunosuppressant in the indicationsdescribed above. For example, it has been possible to demonstrate thatit was possible for antibodies against the CD40 ligand (CD154) oragainst CD3 to prevent or reduce the rejection of transplants. Such(modified) RNAs according to the invention which encode an antibody, thecoded antibodies of which can bind to surface antigens or generally tosurface factors of cells, such as e.g. MHC class I molecules, MHC classII molecules, T cell receptors, LMP2 molecules, LMP7 molecules, CD1,CD2, CD3, CD4, CD8, CD11, CD28, CD30, CD31, CD40, CD50, CD54, CD56,CD58, CD80, CD86, CD95, CD153, CD154, CD178, CD3=TCR (T cell receptor)etc. are therefore preferably employed for use as immunosuppressants.

In this connection, the invention also additionally provides the use ofan antibody-coding (modified) RNA according to the invention or of thepharmaceutical composition described here for therapeutic use forexpansion of (certain) cells in vitro or in vivo. For example, CD4- andCD25-positive cells and regulatory T cells can be stimulated toexpansion by expression of the superantagonistic CD28 antibody.Regulatory T cells which can be multiplied by expression of thesuperantagonistic CD28 antibody play a role above all in autoimmunediseases (Beyersdorf N, Hanke T, Kerkau T, Hunig T. Superagonisticanti-CD28 antibodies: potent activators of regulatory T cells for thetherapy of autoimmune diseases. Ann Rheum Dis. 2005 November; 64).

An antibody-coding (modified) RNA according to the invention or thepharmaceutical composition described here can likewise be used onrheumatoid arthritis for prevention of inflammation reactions byantibodies against e.g. TNFα or other factors exacerbating the undesiredimmune response against e.g. the patients' proteins, as for thetreatment of autoimmune diseases.

A (modified) RNA according to the invention which encodes anti-CD18antibodies or the pharmaceutical composition described here or thepassive vaccine according to the invention can furthermore also be usedfor reduction of inflammations by inhibition of leukocytes, e.g. in theabovementioned indications.

The present invention furthermore also provides a method for treatmentand/or prevention of the abovementioned diseases and, respectively, for(preventive) passive immunization against the abovementioned diseases,which comprises administration of the pharmaceutical compositionaccording to the invention described, the passive vaccine according tothe invention or, respectively, the RNA according to the invention to apatient, in particular a human. Such methods also relate to treatment ofindications which are connected with the intra- and extracellularprocesses described above, with neutralization functions of antibodies,the abovementioned inhibition of certain (cell) functions by antibodiesetc.

The present invention also provides an in vitro transcription method forthe preparation of an antibody-coding (modified) RNA, comprising thefollowing steps:

-   a) provision of a nucleic acid, in particular a cDNA, which codes    for an antibody as described above;-   b) addition of the nucleic acid, in particular a cDNA, which codes    for an antibody to an in vitro transcription medium comprising an    RNA polymerase, a suitable buffer, a nucleic acid mix comprising one    or more optionally modified nucleotides as described above in    exchange for one or more of the naturally occurring nucleotides A,    G, C or U, and optionally one or more naturally occurring    nucleotides A, G, C or U, if not all the naturally occurring    nucleotides A, G, C or U are to be exchanged, and optionally an    RNase inhibitor;-   c) incubation of the nucleic acid, in particular a cDNA, which codes    for an antibody in the in vitro transcription medium and in vitro    transcription of the nucleic acid to give an antibody-coding    optionally modified RNA according to the invention;-   d) optionally purification of the antibody-coding (modified) RNA    according to the invention and removal of the non-incorporated    nucleotides from the in vitro transcription medium.

A nucleic acid as described in step a) of the in vitro transcriptionmethod according to the invention can be any nucleic acid as describedhere (for example single- or double-stranded DNA, cDNA etc.) whichencodes an antibody as described here. DNA sequences, e.g. genomic DNAor fragments thereof, or plasmids which encode an antibody as describedhere, preferably in linearized form, are typically employed for this.The in vitro transcription can conventionally be carried out using avector which has an RNA polymerase binding site. Any (expression)vectors known in the prior art, e.g. commercially obtainable(expression) vectors, can be used for this. Preferred (expression)vectors are, for example, those which have an SP6 or a T7 or T3 bindingsite upstream and/or downstream of the cloning site. The nucleic acidsequences used can thus be transcribed later as desired, depending onthe RNA polymerase chosen. A nucleic acid sequence which is used for thein vitro transcription and codes for an antibody as described here istypically cloned into the (expression) vector, e.g. via a multiplecloning site of the vector used. Before the transcription, the(expression) vector is typically cleaved with restriction enzymes at thesite at which the future 3′ end of the RNA is to be found, using asuitable restriction enzyme, and the fragment is purified. This excludesthe transcribed RNA from containing vector sequences, and an RNAtranscript of defined length is obtained. In this context, preferably norestriction enzymes which generate overhanging ends (such as e.g. AatII, Apa I, Ban II, Bgl I, Bsp 1286, BstX I, Cfo I, Hae II, HgiA I, HhaI, Kpn I, Pst I, Pvu I, Sac I, Sac II, Sfi I, Sph I etc.) are used.Should such restriction enzymes nevertheless be used, the overhanging 3′end is preferably filled up, e.g. with Klenow or T4 DNA polymerase.

As an alternative for step a) the nucleic acid can also be prepared as atranscription template by a polymerase chain reaction (PCR). For this,one of the primers used typically contains the sequence of an RNApolymerase binding site. Furthermore, the 5′ end of the primer usedpreferably contains an extension of about 10-50 further nucleotides,more preferably of from 15 to 30 further nucleotides and most preferablyof about 20 nucleotides.

Before the in vitro transcription, the nucleic acid, e.g. the DNA orcDNA, template is typically purified and freed from RNase in order toensure a high yield. Purification can be carried out with the aid of anymethod known in the prior art, for example using a caesium chloridegradient, ion exchange methods or by purification via agarose gelelectrophoresis.

According to method step b), the nucleic acid (used as the transcriptiontemplate) is added to an in vitro transcription medium. A suitable invitro transcription medium initially comprises a nucleic acid asprovided under step a), for example about 0.1-10 preferably about 1-5more preferably 2.5 μg and most preferably about 1 μg of such a nucleicacid. A suitable in vitro transcription medium furthermore optionallycomprises a reducing agent, e.g. DTT, more preferably about 1-20 μl 50mM DTT, even more preferably about 5 μl 50 mM DTT. The in vitrotranscription medium furthermore comprises nucleotides, e.g. anucleotide mix, in the case of the present invention comprising amixture of (modified) nucleotides as defined here (typically about0.1-10 mM per nucleotide, preferably 0.1 to 1 mM per nucleotide(preferably about 4 mM in total)) and optionally non-modifiednucleotides. If modified nucleotides as defined here (about 1 mM pernucleotide, preferably about 4 mM in total), e.g. pseudouridine5′-triphosphate, 5-methylcytidine 5′-triphosphate etc., are employed,they are typically added in an amount such that the modified orbase-modified nucleotides is completely replaced by the naturalnucleotide. However, it is also possible to employ mixtures of one ormore modified or base-modified nucleotides and one or more naturallyoccurring nucleotides instead of a particular nucleotide, i.e. it isthus possible to employ one or more modified nucleotides as describedabove in exchange for one or more of the naturally occurring nucleotidesA, G, C or U, and optionally additionally one or more naturallyoccurring nucleotides A, G, C or U, if not all the naturally occurringnucleotides A, G, C or U are to be exchanged. Conversely, it is alsopossible to use only natural nucleotides. By selective addition of thedesired nucleotide to the in vitro transcription medium the content,i.e. the occurrence and the amount, of the desired modification ofnucleotides in the transcribed antibody-coding (modified) RNA sequencecan therefore be controlled. A suitable in vitro transcription mediumlikewise comprises an RNA polymerase, e.g. T7 RNA polymerase (forexample T7-Opti mRNA Kit, CureVac, Tubingen, Germany), T3 RNA polymeraseor SP6, typically about 10 to 500 U, preferably about 25 to 250 U, morepreferably about 50 to 150 U, and most preferably about 100 U of RNApolymerase. The in vitro transcription medium is furthermore preferablykept free from RNase in order to avoid degradation of the transcribedRNA. A suitable in vitro transcription medium therefore optionallyadditionally comprises an RNase inhibitor.

The nucleic acid is incubated in the in vitro transcription medium in astep c) and is transcribed to an antibody-coding (modified) RNA. Theincubation times are typically about 30 to 240 minutes, preferably about40 to 120 minutes and most preferably about 90 minutes. The incubationtemperatures are typically about 30-45° C., preferably 37-42° C. Theincubation temperature depends on the RNA polymerase used, e.g. for T7RNA polymerase it is about 37° C. The RNA obtained by the transcriptionis preferably an mRNA. The yields obtained in the in vitro transcriptionare, for the stated starting amounts employed in step b), typically inthe region of about 30 μg of RNA per μg of template DNA used. In thecontext of the present invention, the yields obtained in the in vitrotranscription can be increased by linear up scaling. For this, thestated starting amounts employed in step b) are preferably increasedaccording to the yields required, e.g. by a multiplication factor of 5,10, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000 etc.

After the incubation, a purification of the transcribed antibody-coding(modified) RNA can optionally take place in step d) of the in vitrotranscription method according to the invention. Any suitable methodknown in the prior art, e.g. chromatographic purification methods, e.g.affinity chromatography, gel filtration etc., can be used for this. Bythe purification, non-incorporated, i.e. excess nucleotides and templateDNA can be removed from the in vitro transcription medium and a clean(modified) RNA can be obtained. For example, after the transcription thereaction mixture containing the transcribed RNA can typically bedigested with DNase in order to remove the DNA template still containedin the reaction mixture. The transcribed RNA can be subsequently oralternatively precipitated with LiCl. Purification of the transcribedRNA can then take place via IP RP-HPLC. This renders it possible inparticular to separate longer and shorter fragments from one anothereffectively.

Preferably, in this context the purification takes place via a methodfor purification of RNA on a preparative scale, which is distinguishedin that the RNA is purified by means of HPLC using a porous reversephase as the stationary phase (PURE Messenger). For example, for thepurification in step d) of the in vitro method according to theinvention, a reverse phase can be employed as the stationary phase forthe HPLC purification. For the chromatography with reverse phases, anon-polar compound typically serves as stationary phase, and a polarsolvent, such as mixtures of water, which is usually employed in theform of buffers, with acetonitrile and/or methanol, serves as the mobilephase for the elution. Preferably, the porous reverse phase has aparticle size of 8.0±2 μm, preferably ±1 μm, more preferably +/−0.5 μm.The reverse phase material can be in the form of beads. The purificationcan be carried out in a particularly favourable manner with a porousreverse phase having this particle size, optionally in the form ofbeads, particularly good separation results being obtained. The reversephase employed is preferably porous since with stationary reverse phaseswhich are not porous, such as are described e.g. by Azarani A. andHecker K. H., pressures which are too high are built up, so thatpreparative purification of the RNA is possible, if at all, only withgreat difficulty. The reverse phase preferably has a pore size of from200 {acute over (Å)} to 5,000 {acute over (Å)}, in particular a poresize of from 300 {acute over (Å)} to 4,000 {acute over (Å)}.Particularly preferred pore sizes for the reverse phases are 200-400{acute over (Å)}, 800-1,200 {acute over (Å)} and 3,500-4,500 {acute over(Å)}. With a reverse phase having these pore sizes, particularly goodresults are achieved in respect of the purification of the RNA inprocess step d). The material for the reverse phase is preferably apolystyrene-divinylbenzene, and non-alkylatedpolystyrene-divinylbenzenes can be employed in particular. Stationaryphases with polystyrene-divinylbenzene are known per se. For thepurification in method step d), the polystyrene-divinylbenzenes whichare known per se and already employed for HPLC methods and arecommercially obtainable can be used. A non-alkylated porouspolystyrene-divinylbenzene which in particular has a particle size of8.0±0.5 μm and a pore size of 250-300 {acute over (Å)}, 900-1,100 {acuteover (Å)} or 3,500-4,500 {acute over (Å)} is very particularlypreferably used for the purification in method step d). The advantagesdescribed above can be achieved in a particularly favourable manner withthis material for the reverse phases. The HPLC purification can becarried out by the ion pair method, an ion having a positive chargebeing added to the mobile phase as a counter-ion to the negativelycharged RNA. An ion pair having a lipophilic character, which is sloweddown by the non-polar stationary phase of the reverse phase system, isformed in this manner. In practices, the precise conditions for the ionpair method must be worked out empirically for each concrete separationproblem. The size of the counter-ion, its concentration and the pH ofthe solution contribute greatly towards the result of the separation. Ina favourable manner, alkylammonium salts, such as triethylammoniumacetate and/or tetraalkylammonium compounds, such as tetrabutylammonium,are added to the mobile phase. Preferably, 0.1 M triethylammoniumacetate is added and the pH is adjusted to about 7. The choice of mobilephase depends on the nature of the desired separation. This means thatthe mobile phase found for a specific separation, such as can be known,for example, from the prior art, cannot be transferred readily toanother separation problem with adequate prospect of success. The idealelution conditions, in particular the mobile phase used, must bedetermined for each separation problem by empirical experiments. Amixture of an aqueous solvent and an organic solvent can be employed asthe mobile phase for elution of the RNA by the HPLC method. In thiscontext, it is favourable if a buffer which has, in particular, a pH ofabout 7, for example 6.5-7.5, e.g. 7.0, is used as the aqueous solvent;preferably, the buffer triethylammonium acetate is used, particularlypreferably a 0.1 M triethylammonium acetate buffer which, as describedabove, also acts as a counter-ion to the RNA in the ion pair method. Theorganic solvent employed in the mobile phase can be acetonitrile,methanol or a mixture of these two, very particularly preferablyacetonitrile. The purification of the RNA in method step d) using anHPLC method as described is carried out in a particularly favourablemanner with these organic solvents. The mobile phase is particularlypreferably a mixture of 0.1 M triethylammonium acetate, pH 7, andacetonitrile. It has emerged to be likewise particularly favourable ifthe mobile phase contains 5.0 vol. % to 20.0 vol. % of organic solvent,based on the mobile phase, and the remainder to make up 100 vol. % isthe aqueous solvent. It is very particularly favourable for the methodaccording to the invention if the mobile phase contains 9.5 vol. % to14.5 vol. % of organic solvent, based on the mobile phase, and theremainder to make up 100 vol. % is the aqueous solvent. Elution of theRNA can subsequently be carried out isocratically or by means of agradient separation. In the case of an isocratic separation, elution ofthe RNA is carried out with a single eluting agent or a mixture ofseveral eluting agents which remains constant, it being possible for thesolvents described above in detail to be employed as the eluting agent.

The present invention also provides an in vitro transcription andtranslation method for expression of an antibody, comprising thefollowing steps:

-   a) provision of a nucleic acid, in particular a cDNA, which encodes    an antibody as described above;-   b) addition of the nucleic acid to an in vitro transcription medium    comprising an RNA polymerase, a suitable buffer, a nucleic acid mix    comprising one or more (modified) nucleotides as described above in    exchange for one or more of the naturally occurring nucleotides A,    G, C or U, and optionally one or more naturally occurring    nucleotides A, G, C or U, if not all the naturally occurring    nucleotides A, G, C or U are to be exchanged, and optionally an    RNase inhibitor;-   c) incubation of the nucleic acid, in particular a cDNA, in the in    vitro transcription medium and in vitro transcription of the nucleic    acid to give an antibody-coding (modified) RNA according to the    invention;-   d) optionally purification of the antibody-coding (modified) RNA    according to the invention and removal of the non-incorporated    nucleotides from the in vitro transcription medium;-   e) addition of the (modified) RNA obtained in step c) (and    optionally in step d) to an in vitro translation medium;-   f) incubation of the (modified) RNA in the in vitro translation    medium and in vitro translation of the antibody coded by the    (modified) RNA;-   g) optionally purification of the antibody translated in step f).

Steps a), b), c) and d) of the in vitro transcription and translationmethod according to the invention for expression of an antibody areidentical to steps a), b), c) and d) of the in vitro transcriptionmethod according to the invention described here.

In step e) of the in vitro transcription and translation methodaccording to the invention for expression of an antibody, the (modified)RNA transcribed in step c) (and optionally purified in step d) is addedto a suitable in vitro translation medium. A suitable in vitrotranslation medium comprises, for example, reticulocyte lysate, wheatgerm extract etc. Such a medium conventionally furthermore comprises anamino acid mix. The amino acid mix typically comprises (all) naturallyoccurring amino acids and optionally modified amino acids, e.g.³⁵S-methionine (for example for monitoring the translation efficiencyvia autoradiography). A suitable in vitro translation medium furthermorecomprises a reaction buffer. In vitro translation media are described,for example, in Krieg and Melton (1987) (P. A. Krieg and D. A. Melton(1987) In vitro RNA synthesis with SP6 RNA polymerase; Methods Enzymol155:397-415), the disclosure content of which in this respect isincluded in its full scope in the present invention.

In a step f) of the in vitro transcription and translation methodaccording to the invention for expression of an antibody, the (modified)nucleic acid is incubated in the in vitro translation medium and theantibody coded by the (modified) nucleic acid is translated in vitro.The incubation typically lasts about 30 to 240 minutes, preferably about40 to 120 minutes and most preferably about 90 minutes. The incubationtemperature is typically in a range of about 20-40° C., preferably about25 to 35° C. and most preferably about 30° C.

Steps b) to f) of the in vitro transcription and translation methodaccording to the invention for expression of an antibody or individualsteps of steps b) to f) can be combined with one another, i.e. can becarried out together. In this context, all the necessary components arepreferably added to the reaction medium together at the start orsuccessively during the reaction in accordance with the sequence of thesteps b) to f) described.

The translated antibody obtained in step f) can be purified in anoptional step g). A purification can be carried out with methods whichare known to a person skilled in the art from the prior art, e.g.chromatography, such as, for example, affinity chromatography (HPLC,FPLC, etc.), ion exchange chromatography, gel chromatography, sizeexclusion chromatography, gas chromatography, or antibody detection, orbiophysical methods, such as e.g. NMR analyses, etc. (see e.g. Maniatiset al. (2001) supra). Chromatography methods, including affinitychromatography methods, can employ tags in a suitable manner for thepurification, as described above, e.g. a hexahistidine tag (SEQ ID NO:59) (His tag, polyhistidine tag), a streptavidin tag (Strep tag), an SBPtag (streptavidin-binding tag), a GST (glutathione S transferase) tagetc. The purification can furthermore be carried out via an antibodyepitope (antibody-binding tag), e.g. a Myc tag, an Swa11 epitope, a FLAGtag, an HA tag etc., i.e. via recognition of the epitope via acorresponding (immobilized) antibody. The purification can likewise becarried out via the immobilized substrate of the specific antibody, i.e.by binding of the antibody to an immobilized antigen which is recognizedand, respectively, bound specifically by the translated antibody.

The present invention also provides an in vitro transcription andtranslation method for expression of an antibody in a host cell,comprising the following steps:

-   a) provision of a nucleic acid, in particular a cDNA, which encodes    an antibody as described above;-   b) addition of the nucleic acid to an in vitro transcription medium    comprising an RNA polymerase, a suitable buffer, one or more    (modified) nucleotides as described above in exchange for one or    more of the naturally occurring nucleotides A, G, C or U and    optionally one or more naturally occurring nucleotides A, G, C or U,    if not all the naturally occurring nucleotides A, G, C or U are to    be exchanged;-   c) incubation of the nucleic acid, in particular a cDNA, in the in    vitro transcription medium and in vitro transcription of the nucleic    acid to give an antibody-coding (modified) RNA according to the    invention;    -   d) optionally purification of the antibody-coding (modified) RNA        according to the invention and removal of the non-incorporated        nucleotides from the in vitro transcription medium;-   e′) transfection of the (modified) RNA obtained in step c) (and    optionally d)) into a host cell;-   f′) incubation of the (modified) nucleic acid in the host cell and    translation of the antibody coded by the (modified) RNA in the host    cell;-   g′) optionally isolation and/or purification of the antibody    translated in step f′).

Steps a), b), c) and d) of the in vitro transcription and translationmethod according to the invention for expression of an antibody in ahost cell are identical to steps a), b), c) and d) of the in vitrotranscription method according to the invention described here and ofthe in vitro transcription and translation method according to theinvention described here for expression of an antibody.

According to step e′) of the in vitro transcription and translationmethod according to the invention, transfection of the (modified) RNAobtained in step c) (and optionally in step d)) into a host cell iscarried out. The transfection is in general carried out via transfectionmethods known in the prior art (see, for example, Maniatis et al. (2001)Molecular Cloning: A laboratory manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). Suitable transfection methods in thecontext of the present invention include, without being limited thereto,e.g. electroporation methods, including modified electroporation methods(e.g. nucleofection), calcium phosphate methods, e.g. the calciumcoprecipitation method, the DEAE-dextran method, the lipofection method,e.g. the transferrin-mediated lipofection method, polyprenetransfection, particle bombardment, nanoplexes, e.g. PLGA, polyplexes,e.g. PEI, protoplast fusion and the microinjection method, thelipofection method having emerged in particular as a suitable method. Inthis context, the (modified) RNA according to the invention can be inthe naked or complexed form, as described above for the (modified) RNAaccording to the invention.

In connection with the present invention and with step e′) of the invitro transcription and translation method according to the inventionfor expression of an antibody in a host cell, a (suitable) host cellincludes any cell which allows expression of the antibody coded by the(modified) RNA according to the invention, preferably any culturedeukaryotic cell (e.g. yeast cells, plant cells, animal cells and humancells) or prokaryotic cell (e.g. bacteria cells etc.). Cells ofmulticellular organisms are preferably chosen for expression of theantibody coded by the (modified) RNA according to the invention ifposttranslational modifications, e.g. glycosylation of the codedprotein, are necessary (N- and/or O-coupled). In contrast to prokaryoticcells, such (higher) eukaryotic cells render possible posttranslationalmodification of the protein synthesized. The person skilled in the artknows a large number of such higher eukaryotic cells or cell lines, e.g.293T (embryonal kidney cell line), HeLa (human cervix carcinoma cells),CHO (cells from the ovaries of the Chinese hamster) and further celllines, including such cells and cell lines developed for laboratorypurposes, such as, for example, hTERT-MSC, HEK293, Sf9 or COS cells.Suitable eukaryotic cells furthermore include cells or cell lines whichare impaired by diseases or infections, e.g. cancer cells, in particularcancer cells of any of the types of cancer mentioned here in thedescription, cells impaired by HIV, and/or cells of the immune system orof the central nervous system (CNS). Human cells or animal cells, e.g.of animals as mentioned here, are particularly preferred as eukaryoticcells. Suitable host cells can likewise be derived from eukaryoticmicroorganisms, such as yeast, e.g. Saccharomyces cerevisiae (Stinchcombet al., Nature, 282:39, (1997)), Schizosaccharomyces pombe, Candida,Pichia, and filamentous fungi of the genera Aspergillus, Penicillium,etc. Suitable host cells likewise include prokaryotic cells, such ase.g. bacteria cells, e.g. from Escherichia coli or from bacteria of thegenera Bacillus, Lactococcus, Lactobacillus, Pseudomonas, Streptomyces,Streptococcus, Staphylococcus, preferably E. coli, etc.

In step f′) of the in vitro transcription and translation methodaccording to the invention for expression of an antibody in a host cell,incubation of the (modified) RNA in the host cell and translation of theantibody coded by the (modified) RNA in the host cell are carried out.Expression mechanisms intrinsic to the host cell are preferably used forthis, e.g. by translation of the RNA in the host cell via ribosomes andtRNAs. The incubation temperatures used in this context depend on theparticular host cell systems used.

In an optional step g′), the translated antibody obtained in step f′)can be isolated and/or purified. In this context, an isolation of thetranslated (expressed) antibody typically comprises separating off theantibody from reaction constituents, and can be carried out by methodswhich are known to a person skilled in the art, for example by celllysis, breakdown by ultrasound, or similar methods, including theabovementioned methods. A purification can therefore also be carried outby methods as described for step e) of the in vitro transcription andtranslation method according to the invention for expression of anantibody.

For purification of (recombinant) antibodies from a host cell in stepg′) of the method described above, a different choice of the host cellsdescribed above is necessary, depending on the use. Thus, the productionof recombinant antibodies in E. coli typically is possible to only alimited extent, since the antibodies coded by a (modified) RNA accordingto the invention are very complex, require complicated foldingmechanisms and are conventionally modified posttranslationally for usein multicellular organisms or beings. These mechanisms conventionallycannot be implemented in the cytoplasm of E. coli. Periplasmicproduction in E. coli, in which correct folding and modification of theantibody fragments is possible, can therefore be used. In this context,the purification usually requires an involved breakdown of the bacteriaand a difficult separating off of all the bacterial constituents whichcan act as endotoxins during a therapeutic use. To bypass thesepurification problems, expression systems for yeasts, insect cells,mammalian cells and plants can be employed according to the invention insuch cases, the production preferably being carried out in suitablemammalian cells, such as e.g. hamster cells (CHO), as described here.

Regardless of steps (a) to (d), the antibody coded by the (modified) RNAaccording to the invention can also be expressed by an in vitrotranslation method of steps (e′) to (g′), which is also subject matterof the present invention as such.

The present invention also provides an in vitro transcription and invivo translation method for expression of an antibody in an organism,comprising the following steps:

-   a) provision of a nucleic acid, in particular a cDNA, which encodes    an antibody as described above;-   b) addition of the nucleic acid to an in vitro transcription medium    comprising an RNA polymerase, a suitable buffer, a nucleic acid mix    comprising one or more (modified) nucleotides as described above in    exchange for one or more of the naturally occurring nucleotides A,    G, C or U, and optionally one or more naturally occurring    nucleotides A, G, C or U, if not all the naturally occurring    nucleotides A, G, C or U are to be exchanged, and optionally an    RNase inhibitor;-   c) incubation of the nucleic acid, in particular a cDNA, in the in    vitro transcription medium and in vitro transcription of the nucleic    acid to give a (modified) RNA according to the invention as    described here;-   d) optionally purification and removal of the non-incorporated    nucleotides from the in vitro transcription medium;-   e″) transfection of the (modified) RNA obtained in step c) (and    optionally in step d)) into a host cell and transplanting of the    transfected host cell into an organism;-   f″) translation of the antibody coded by the (modified) RNA in the    organism.

Steps a), b), c) and d) of the in vitro transcription and in vivotranslation method according to the invention for expression of anantibody in an organism are identical to steps a), b), c) and d) of thein vitro transcription method according to the invention described here,of the in vitro transcription and translation method according to theinvention described here for expression of an antibody and of the invitro transcription and translation method according to the inventiondescribed here for expression of an antibody in a host cell.

Host cells in the context of the present invention, and in particular instep e″), can also include autologous cells, i.e. cells which are takenfrom a patient and returned again (endogenous cells). Such autologouscells reduce the risk of rejection by the immune system in the case ofin vivo uses. In the case of autologous cells, (healthy or diseased)cells from the affected body regions/organs of the patient arepreferably employed. Transfection methods are preferably those asdescribed above for step e). In step e″), transplanting of the host cellinto an organism is carried out, additionally to step e). An organism ora being in connection with the present invention typically meansmammals, i.e. animals, including cattle, pig, dog, cat, donkey, monkey,including rodents, e.g. mouse, hamster, rabbit etc., and humans. As analternative to step e″) and f′), the isolation and/or purification canbe carried out according to steps f)/f′) and/or g)/g′) and thetranslated (therapeutically active) protein can be administeredsubsequently to the being. The administration can be carried out asdescribed for pharmaceutical compositions.

In step f″), translation of the antibody coded by the (modified) RNA iscarried out in the organism. In this context, the translation ispreferably carried out by means of systems specific to the host cell,depending on the host cell used.

Regardless of steps (a) to (d), the transcribed (modified) RNA accordingto the invention can also be expressed by an in vitro translation methodof steps (e″) to (g″), which is also subject matter of the presentinvention as such.

As an alternative to the methods described above, according to aparticularly preferred embodiment in a further step e′″) a (modified)RNA according to the invention transcribed according to steps a) to d)can be administered directly into the organism, e.g. human, e.g. byadministration of the naked or complexed RNA according to the invention,for example using the transfection methods described above, optionallyusing certain stabilizing factors described here. In this context, afteruptake the RNA is preferably transported into the cells, e.g. withlocalization or signal sequences as described here, and preferablytranslated into the coded antibody in the cells.

Advantages of the Invention

The present invention describes in particular an antibody-coding RNAaccording to the invention. This can be modified or non-modified, wherethe definition of “modification” is to be understood in the broadestsense. A native RNA covalently bonded to another group, for example alipid or a sugar residue, is modified in the context of this invention.An RNA which contains non-natively occurring constituents, for examplenon-native nucleotides, or an RNA which is modified with respect to itsprecursor by exchange of nucleotides, regardless of whether these arenative or non-native, is also modified in the context of the invention.The great advantage of such RNAs is that these do not have the negativeactions of DNA transfections (with stable incorporation into thegenome). In the case of modified antibody-coding RNAs, the limitedstability of the RNA coding for the antibodies or antibody fragments ismoreover improved. According to the invention, after administration topatients, in particular mammals, above all humans, the antibodies aretherefore thus expressed in vivo for only an estimatable time beyond thetreatment and therefore do not lead to harmful effects. In contrast, theconventional intrabody DNAs can be integrated into the genome in astable manner or at least expressed persistently, which can lead touncontrollable events. The great advantage compared with administrationof monoclonal antibodies in vivo is furthermore that with the use of anantibody-coding (modified) RNA as described here, no antibodies have tobe prepared and purified in an involved manner and they are thereforeconsiderably less expensive to prepare. The most essential advantage ofthe present invention is, however, that intracellularly expressedproteins can also be achieved with the antibodies coded by (modified)RNAs according to the invention, which is not possible with monoclonalantibodies known hitherto from the prior art.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures and examples are intended to explain in moredetail and illustrate the above description, without being limitedthereto.

FIG. 1 illustrates the structure of an IgG antibody. IgG antibodies arebuilt up from in each case two identical light and two heavy proteinchains which are bonded to one another via disulfide bridges. The lightchain comprises the N-terminal variable domain V_(L) and the C-terminalconstant domain C_(L). The heavy chain of an IgG antibody can be dividedinto an N-terminal variable domain V_(H) and three constant domainsC_(H)1, C_(H)2 and C_(H)3.

FIGS. 2A-D show the gene cluster for the light and the heavy chains ofan antibody:

-   -   (A): Gene cluster for the light chain κ.    -   (B): Gene cluster for the light chain λ.    -   (C): and (D): Gene cluster for the heavy chain.    -   In this context, the variable region of a heavy chain is        composed of three different gene segments. In addition to the V        and J segments, additional D segments are also found here. The        V_(H), D_(H) and J_(H) segments can likewise be combined with        one another virtually as desired to form the variable region of        the heavy chain.

FIG. 3 illustrates in the form of a diagram the differences in the lightand heavy chains of murine (i.e. obtained in the mouse host organism),chimeric, humanized and human antibodies.

FIG. 4 shows an overview of the structure of various antibody fragments.The constituents of the antibody fragments are shown on a dark greybackground.

FIGS. 5A-C show various variants of antibodies and antibody fragments inFIGS. 5A, 5B and 5C:

-   -   (A) shows a diagram of an IgG antibody of two light and two        heavy chains.    -   (B) shows an Fab fragments from the variable and a constant        domain in each case of a light and a heavy chain. The two chains        are bonded to one another via a disulfide bridge.    -   (C) shows an scFv fragment from the variable domain of the light        and the heavy chain, which are bonded to one another via an        artificial polypeptide linker.

FIG. 6 shows a presentation of an antibody-coding (modified) RNAaccording to the invention as an expression construct. In this:

-   -   V_(H)=variable domain of the heavy chain;    -   C_(H)=constant domain of the heavy chain;    -   V_(L)=variable domain of the light chain;    -   C_(L)=constant domain of the light chain;    -   SIRES=internal ribosomal entry site (IRES, superIRES)    -   muag=mutated form of the 3′ UTR of the alpha-globin gene; and    -   A70C30=polyA-polyC tail.

FIG. 7 shows a diagram of the detection of an antibody coded by an RNAaccording to the invention by means of ELISA on the example of theantigen Her2.

FIG. 8 shows the wild-type DNA sequence of the heavy chain of theantibody rituximab (=Rituxan, MabThera) (wild-type: GC content: 56.5%,length: 1,344) (SEQ ID NO: 1).

FIG. 9 shows the GC-optimized DNA sequence of the heavy chain of theantibody rituximab (=Rituxan, MabThera) (GC content: 65.9%, length:1,344) (SEQ ID NO: 2).

FIG. 10 shows the wild-type DNA sequence of the light chain of theantibody rituximab (=Rituxan, MabThera) (wild-type: GC content: 58.5%,length: 633) (SEQ ID NO: 3).

FIG. 11 shows the GC-optimized DNA sequence of the light chain of theantibody rituximab (=Rituxan, MabThera) (GC content: 67.2%, length: 633)(SEQ ID NO: 4).

FIG. 12 shows the total construct of the GC-optimized DNA sequence ofthe antibody rituximab (=Rituxan, MabThera) with the light and heavychains (SEQ ID NO: 5). The total construct contains the followingsequences and cleavage sites (see also alternative cleavage sites ofFIG. 25, SEQ ID No. 51):

AAGCTT HindIII

(SEQ ID NO: 60) CATCATCATCATCATCAT His tagSignal peptide, HLA-A*0201: GC-rich (SEQ ID NO: 61) ATGGCCGTGATGGCGCCGCG- GACCCTGGTCCTCCTGCTGAGCGGCGCCCTCGCCCTGACGCAGAC-CTGGGCCGGG.

-   -   The coding region of the heavy chain sequence starts with the        signal peptide as given above (italic). This region is G/C        enriched as well. The subsequent sequence starting with CAG        represents the actual antibody coding sequence (see FIG. 9) for        the heavy chain, which ends with AAG and is followed by the        above described His tag sequence. Finally, the open reading        frame for the heavy chain ends with the stop codon TGA (        ) The coding region for the light chain sequence starts 3′        upstream with the signal peptide's ATG as given above followed        by the light chain's coding region for the light chain starting        with CAG running to the stop codon TGA (        ) (see FIG. 11). Both coding regions for the light and the heavy        chain are separated by an IRES element (        ) The inventive RNA coded by the construct given in FIG. 12 may        or may not contain a (His) tag sequence and may contain a signal        peptide sequence different from the above peptide sequence or        may even have no signal peptide sequence. Accordingly, the        inventive RNA molecule contains preferably the coding region        (with or without a signal peptide sequence at its beginning) of        the heavy and/or the light chain (e.g. as shown in FIG. 12),        preferably in combination with at least one ribosomal entry        site.

FIG. 13 shows the wild-type DNA sequence of the heavy chain of theantibody cetuximab (=Erbitux) (wild-type: GC content: 56.8%, length:1,359) (SEQ ID NO: 6).

FIG. 14 shows the GC-optimized DNA sequence of the heavy chain of theantibody cetuximab (=Erbitux) (GC content: 65.9%, length: 1,359) (SEQ IDNO: 7).

FIG. 15 shows the wild-type DNA sequence of the light chain of theantibody cetuximab (=Erbitux) (wild-type: GC content: 58.2%, length:642) (SEQ ID NO: 8).

FIG. 16 shows the GC-optimized DNA sequence of the light chain of theantibody cetuximab (=Erbitux) (GC content: 65.7%, length: 642) (SEQ IDNO: 9).

FIG. 17 shows the total construct of the GC-optimized DNA sequence ofthe antibody cetuximab (=Erbitux) with the light and heavy chains (SEQID NO: 10). The total construct contains the following sequences andcleavage sites (see also alternative cleavage sites of FIG. 26, SEQ IDNo 52):

AAGCTT HindIII

(SEQ ID NO: 60) CATCATCATCATCATCAT His tagSignal peptide, HLA-A*0201: GC-rich (SEQ ID NO: 61) ATGGCCGTGATGGCGCCGCG- GACCCTGGTCCTCCTGCTGAGCGGCGCCCTCGCCCTGACGCAGAC-CTGGGCCGGG.

-   -   The coding region of the heavy chain sequence starts with the        signal peptide as given above (italic). This region is G/C        enriched as well. The subsequent sequence starting with CAG        represents the actual antibody coding sequence (see FIG. 14) for        the heavy chain, which ends with AAG and is followed by the        above described His tag sequence. Finally, the open reading        frame for the heavy chain ends with the stop codon TGA (        ). The coding region for the light chain sequence starts 3′        upstream with the signal peptide's ATG as given above followed        by the light chain's coding region for the light chain starting        with GAC running to the stop codon TGA (        ) (see FIG. 16). Both coding regions for the light and the heavy        chain are separated by an IRES element (        ) The inventive RNA coded by the construct given in FIG. 17 may        or may not contain a (His) tag sequence and may contain a signal        peptide sequence different from the above peptide sequence or        may even have no signal peptide sequence. Accordingly, the        inventive RNA molecule contains preferably the coding region        (with or without a signal peptide sequence at its beginning) of        the heavy and/or the light chain (e.g. as shown in FIG. 17),        preferably in combination with at least one ribosomal entry        site.

FIG. 18 shows the wild-type DNA sequence of the heavy chain of theantibody trastuzumab (=Herceptin) (wild-type: GC content: 57.8%, length:1,356) (SEQ ID NO: 11).

FIG. 19 shows the GC-optimized DNA sequence of the heavy chain of theantibody trastuzumab (=Herceptin) (GC content: 67.0%, length: 1,356)(SEQ ID NO: 12).

FIG. 20 shows the wild-type DNA sequence of the light chain of theantibody trastuzumab (=Herceptin) (wild-type: GC content: 56.9%, length:645) (SEQ ID NO: 13).

FIG. 21 shows the GC-optimized DNA sequence of the light chain of theantibody trastuzumab (=Herceptin) (GC content: 66.4%, length: 645) (SEQID NO: 14).

FIG. 22 shows the total construct of the GC-optimized DNA sequence ofthe antibody trastuzumab (=Herceptin) with the light and heavy chains(SEQ ID NO: 15). The total construct contains the following sequencesand cleavage sites (see also alternative cleavage sites of FIG. 27, SEQID No. 53):

AAGCTT HindIII

(SEQ ID NO: 60) CATCATCATCATCATCAT His tagSignal peptide, HLA-A*0201: GC-rich (SEQ ID NO: 61) ATGGCCGTGATGGCGCCGCG- GACCCTGGTCCTCCTGCTGAGCGGCGCCCTCGCCCTGACGCAGAC-CTGGGCCGGG.

-   -   The coding region of the heavy chain sequence starts with the        signal peptide as given above (italic). This region is G/C        enriched as well. The subsequent sequence starting with GAG        represents the actual antibody coding sequence (see FIG. 19) for        the heavy chain, which ends with AAG and is followed by the        above described His tag sequence. Finally, the open reading        frame for the heavy chain ends with the stop codon TGA (        ) The coding region for the light chain sequence starts 3′        upstream with the signal peptide's ATG as given above followed        by the light chain's coding region for the light chain starting        with GAC running to the stop codon TGA (        ) (see FIG. 21). Both coding regions for the light and the heavy        chain are separated by an IRES element (        ) The inventive RNA coded by the construct given in FIG. 22 may        or may not contain a (His) tag sequence and may contain a signal        peptide sequence different from the above peptide sequence or        may even have no signal peptide sequence. Accordingly, the        inventive RNA molecule contains preferably the coding region        (with or without a signal peptide sequence at its beginning) of        the heavy and/or the light chain (e.g. as shown in FIG. 22),        preferably in combination with at least one ribosomal entry        site.

FIG. 23 shows RNA-mediated antibody expression in cell culture. CHO orBHK cells were transfected with 20 μg of antibody-encoding mRNAaccording to the invention which was produced (RNA, G/C enriched, seeabove) or mock-transfected. 24 hours after transfection proteinsynthesis was analysed by Western blotting of cell lysates. Cellsharboured about 0.5 μg of protein as assessed by Western Blot analysis.Each lane represents 10% of total lysate. Humanised antibodies served ascontrol and for a rough estimate of protein levels. The detectionantibody recognises both heavy and light chains; moreover, it shows someunspecific staining with cell lysates (three distinct bands migratingmuch slower than those of the antibodies). A comparison with controlantibodies clearly demonstrates that heavy and light chains wereproduced in equal amounts.

FIGS. 24A-E show that RNA-mediated antibody expression gives rise to afunctional protein (antibody). Functional antibody formation wasaddressed by FACS staining of antigen-expressing target cells. In orderto examine the production of functional antibodies, cell culturesupernatants of RNA-transfected (20 μg of Ab-RNA as defined above inExample 1) cells were collected after 48 to 96 hours. About 8% of totalsupernatant was used to stain target cells expressing the respectiveantigen. Humanised antibodies served as control and for a rough estimateof protein levels. Primary antibody used for cell staining: a) humanisedantibody; b) none; c,d) supernatant from RNA-transfected cellsexpressing the respective antibody; e) supernatant from mock-transfectedCHO cells. Calculations on the basis of the analysis shown in FIG. 24reveal that cells secreted more than 12-15 μg of functional antibodywithin 48-96 hours. Accordingly, the present invention proves that RNAencoding antibodies may enter into cell, may be expressed within thecell and considerable amounts of RNA encoded antibodies are thensecreted by the cell into the surrounding medium/extracellular space.Cell transfection in vivo or in vitro by the inventive RNA may thereforebe used to provide antibodies acting e.g. therapeutically in theextracellular space.

FIG. 25 shows an alternative sequence of the construct of FIG. 12(antibody rituximab), wherein the restriction sites have been modifiedas compared to SEQ ID No. 5 of FIG. 12 (SEQ ID No.: 51). For closerinformation with regard to the description of various sequence elementsit is referred to FIG. 12.

FIG. 26 shows an alternative sequence of the construct of FIG. 17(antibody cetuximab), wherein the restriction sites have been modifiedas compared to SEQ ID No. 10 of FIG. 17 (SEQ ID No.: 52). For closerinformation with regard to the description of various sequence elementsit is referred to FIG. 17.

FIG. 27 shows an alternative sequence of the construct of FIG. 22(antibody trastuzumab), wherein the restriction sites have been modifiedas compared to SEQ ID No. 15 of FIG. 22 (SEQ ID No.: 53). For closerinformation with regard to the description of various sequence elementsit is referred to FIG. 22.

The following examples explain the present invention in more detail,without limiting it.

EXAMPLES 1. Example 1.1 Cell Lines and Cell Culture Conditions Used

The cell lines HeLa (human cervix carcinoma cell line; Her2-positive),HEK293 (human embryonal kidney; Her2-negative) and BHK21 (Syrian hamsterkidney; Her2-negative) were obtained from the DMSZ (Deutsche Sammlungvon Mikroorganismen and Zellkulturen GmbH) in Braunschweig and culturedin RPMI medium enriched with 2 mM L-glutamine (Bio Whittaker) and 10μg/ml streptomycin and 10 U/ml of penicillin at 37° C. under 5% CO₂.

1.2 Preparation of Expression Vectors for Modified RNA SequencesAccording to the Invention

For the production of modified RNA sequences according to the invention,the GC-enriched and translation-optimized DNA sequences which code for aheavy chain and a light chain of an antibody (e.g. cetuximab (Erbitux),trastuzumab (Herceptin) and rituximab (Rituxan), cf. SEQ ID NO: 1-15,where SEQ ID NO: 1, 3, 6, 8, 11 and 13 represent the particular codingsequences which are not GC-optimized of the heavy and the light chainsof these antibodies and SEQ ID NO: 2, 4, 5, 7, 9, 10, 12, 14 and 15represent the coding GC-enriched sequences (see above)) were cloned intothe pCV19 vector (CureVac GmbH) by standard molecular biology methods.To ensure equimolar expression of the two chains, an IRES (internalribosomal entry site) was introduced. The mutated 3′ UTR (untranslatedregion) of the alpha-globin gene and a polyA-polyC tail at the 3′ endserve for additional stabilizing of the mRNA. The signal peptide of theHLA-A*0201 gene is coded for secretion of the antibody expressed. A Histag was additionally introduced for detection of the antibody. FIG. 6shows the expression constructs used.

1.3 Preparation of the G/C-Enriched and Translation-OptimizedAntibody-Coding mRNA

An in vitro transcription was carried out by means of T7 polymerase(T7-Opti mRNA Kit, CureVac, Tubingen, Germany), followed by purificationwith Pure Messenger™ (CureVac, Tubingen, Germany). For this, a DNasedigestion was first carried out, followed by an LiCl precipitation andthereafter an HPLC using a porous reverse phase as the stationary phase(PURE Messenger).

1.4 Detection of RNA-Antibody by Means of Flow Cytometry

1 million cells were transfected with the mRNA according to one of SEQID NO: 5, 10 or 15 (see above), which codes for an antibody as describedabove, by means of electroporation and were then cultured in the mediumfor 16 h. The antibody expressed was detected by means of anFITC-coupled His tag antibody. Alternatively, the secreted antibody fromthe supernatant of transfected cells was added to non-transfected,antigen-expressing cells and, after incubation, detected by the samemethod.

1.5 In Vitro Detection of an Antibody Coded by an RNA According to theInvention by Means of ELISA

A microtitre plate was loaded with a murine antibody (1) against a firstantigen (HER-2). Cell lysate of antigen-expressing cells was then addedto the plate. The antigen was bound here by the murine antigen-specificantibody (1). The supernatant of cells which were transfected with amodified mRNA according to the invention which codes for anHER-2-specific antibody was then added to the microtitre plate. TheHER-2-specific antibody (2) contained in the supernatant likewise bindsto the antibody-bound antigen, the two antibodies recognizing differentdomains of the antigen. For detection of the bound antibody (2),anti-human IgG coupled to horseradish peroxidase (3-HRP) was added, thesubstrate TMB being converted and the result determined photometrically.

1.6 In Vivo Detection of an Antibody Coded by an RNA According to theInvention

An antibody-coding (m)RNA according to the invention as described abovewas injected intradermally or intramuscularly into BALB/c mice. 24 hthereafter, the corresponding tissues were removed and protein extractswere prepared. The expression of the antibody was detected by means ofELISA as described here.

1.7 Detection of an Antibody Coded by an RNA According to the Inventionby Means of Western Blotting

The expressed antibodies from the supernatant of cells which weretransfected with a modified mRNA which codes for an antibody asdescribed above were separated by means of a polyacrylamide gelelectrophoresis and then transferred to a membrane. After incubationwith antiHis tag antibody and a second antibody coupled to horseradishperoxidase, the antibody expressed was detected by means ofchemoluminescence.

1.8 Tumour Model

SKOV-3 cells were injected subcutaneously into BALB/c mice. Within thefollowing 28 days, eight portions of 10 μg of a modified mRNA whichcodes for an antibody as described above were injected into the tailvein of the mice. The tumour growth was monitored over a period of 5weeks.

2. Example 2.1. Cell Lines

RNA-based expression of humanised antibodies was done in either CHO-K1or BHK-21 cells. The tumour cell lines BT-474, A-431 and Raji stronglyexpressing HER2, EGFR and CD20, respectively, were used to recordantibody levels. All cell lines except CHO were maintained in RPMIsupplemented with FCS and glutamine according to the supplier'sinformation. CHO cells were grown in Ham's F12 supplemented with 10%FCS. All cell lines were obtained from the German collection of cellcultures (DSMZ).

2.2. Antibody Expression

Various amounts of antibody-RNA (G/C enriched as defined by FIGS. 12,17, 22, 25, 26, 27) encoding the humanised antibodies Herceptin,Erbitux, and Rituxan, respectively, (see the description given above forExample 1) were transfected into either CHO or BHK cells byelectroporation. Conditions were as follows: 300 V, 450 μF for CHO and300 V, 150 μF for BHK. After transfection, cells were seeded onto24-well cell culture plates at a density of 2-400.000 cells per well.For collection of secreted protein, medium was replaced by 250 μl offresh medium after cell attachment to the plastic surface. Secretedprotein was collected for 24-96 hours and stored at 4° C. In addition,cells were harvested into 50 μl of phosphate buffered saline containing0.5% BSA and broken up by three freeze-thaw cycles. Cell lysates werecleared by centrifugation and stored at −80° C.

2.3. Western Blot Analysis

In order to detect translation of transfected RNA, proteins from eithercell culture supernatants or cell lysates were separated by a 12%SDS-PAGE and blotted onto a nitrocellulose membrane. Humanisedantibodies Herceptin (Roche), Erbitux (Merck KGAA), and Mabthera=Rituxan(Roche) were used as controls. After blotting was completed, membraneswere consecutively incubated with biotinylated goat anti-human IgG(Dianova), streptavidin coupled to horseradish peroxidase (BD), and achemiluminescent substrate (SuperSignal West Pico, Pierce). Staining wasdetected with a Fuji LAS-1000 chemiluminescence camera.

2.4. FACS Analysis

200.000 target cells expressing the respective antigen were incubatedwith either control antibodies (Herceptin, Erbitux, Mabthera) or cellculture supernatants. For detection of bound antibodies, cells werestained with biotinylated goat anti-human IgG (Dianova) and PE-labelledstreptavidin (Invitrogen). Cells were analysed on a FACSCalibur (BD).

What is claimed is:
 1. A method of treating a subject comprisingadministering an effective amount of a pharmaceutical compositioncomprising mRNA encoding a botulism toxin-binding antibody.
 2. Themethod of claim 1, wherein the subject has a botulism infection.
 3. Themethod of claim 1, wherein the pharmaceutical composition isadministered by injection.
 4. The method of claim 1, wherein theantibody comprises a Fab, Fab′, F(ab′)2, Fc, pFc′, Fd, FIT and scFvfragment of an antibody.
 5. The method of claim 1, wherein the antibodycomprises a human antibody or a humanized antibody.
 6. The method ofclaim 1, wherein the mRNA comprises a sequence encoding an antibodyoperably linked to a heterologous secretory signal sequence.
 7. Themethod of claim 1, wherein the composition comprises a mRNA that encodesan antibody light chain and a mRNA that encodes an antibody heavy chain.8. The method of claim 1, wherein the composition comprises a mRNA thatencodes an antibody light chain and an antibody heavy chain, wherein theantibody light chain and an antibody heavy chain coding sequences arelinked by an internal ribosomal entry site (IRES).
 9. The method ofclaim 1, wherein the mRNA comprises a 5′ cap structure.
 10. The methodof claim 1, wherein the mRNA additionally comprises a poly-A tail of 10to 200 adenosine nucleotides.
 11. The method of claim 1, wherein themRNA additionally comprises a poly-C tail of 10 to 200 cytosinenucleotides.
 12. The method of claim 1, wherein the mRNA is modified byintroduction of a non-native nucleotide compared with a native mRNAsequence and/or by covalent coupling of the mRNA with a further chemicalmoiety.
 13. The method of claim 12, wherein the mRNA comprises a G/Ccontent in the antibody coding region which is greater than the G/Ccontent of the coding region of the native mRNA sequence encoding theantibody.
 14. The method of claim 12, wherein the mRNA comprises anantibody coding sequence that is modified, compared with the native mRNAencoding the antibody, such that at least one codon of the native mRNAwhich codes for a tRNA which is relatively rare in the cell is exchangedfor a codon which codes for a tRNA which is relatively frequent in thecell.
 15. The method of claim 12, wherein the mRNA comprises a chemicalmodification relative to a naturally occurring mRNA.
 16. The method ofclaim 12, wherein the mRNA comprises at least a nucleotide that issubstituted with a nucleotide analog selected from the group consistingof: 1-methyl-adenine, 2-methyl-adenine,2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine,N6-isopentenyl-adenine, 2-thio-cytosine, 3-methylcytosine,4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine,1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine,7-methyl-guanine, inosine, 1-methyl-inosine, dihydro-uracil,2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil,5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil,5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil,5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester,5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil,5-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyaceticacid methyl ester, uracil-5-oxyacetic acid (v), pseudouracil,1-methyl-pseudouracil, queosine, β-D-mannosyl-queosine, wybutoxosine,phosphoramidates, phosphorothioates, peptide nucleotides,methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. 17.The method of claim 12, wherein the mRNA modification comprises at leastone base-modified nucleotide chosen from the group consisting of2-amino-6-chloropurine riboside 5′-triphosphate, 2-aminoadenosine5′-triphosphate, 2-thiocytidine 5′-triphosphate, 2-thiouridine5′-triphosphate, 4-thiouridine 5′-triphosphate, 5-aminoallylcytidine5′-triphosphate, 5-aminoallyluridine 5′-triphosphate, 5-bromocytidine5′-triphosphate, 5-bromouridine 5′-triphosphate, 5-iodocytidine5′-triphosphate, 5-iodouridine 5′-triphosphate, 5-methylcytidine5′-triphosphate, 5-methyluridine 5′triphosphate, 6-azacytidine5′-triphosphate, 6-azauridine 5′-triphosphate, 6-chloropurine riboside5′-triphosphate, 7-deazaadenosine 5′-triphosphate, 7-deazaguanosine5′-triphosphate, 8-azaadenosine 5′-triphosphate, 8-azidoadenosine5′-triphosphate, benzimidazole riboside 5′-triphosphate,N1-methyladenosine 5′-triphosphate, N1-methylguanosine 5′-triphosphate,N6-methyladenosine 5′-triphosphate, 06-methylguanosine 5′-triphosphate,pseudouridine 5′-triphosphate, puromycin 5′-triphosphate and xanthosine5′-triphosphate.
 18. The method of claim 17, wherein the base-modifiednucleotide is chosen from the group consisting of: 5-methylcytidine5′-triphosphate, 1-methyl-pseudouracil and pseudouridine5′-triphosphate.
 19. A pharmaceutical composition comprising an isolatedmRNA comprising a coding region encoding at least one antibody variabledomain of a botulism toxin-binding antibody, wherein said coding regionis linked to a heterologous secretory signal sequence.